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WORLD INTELLECTUAL PROPERTY ORGANIZATION
International Bureau

PCT

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification 6 :
G01N

(11) International Publication Number: WO 99/45357

(43) International Publication Date: 10 September 1999 (10.09.99)

(21) International Application Number: PCT/US99/04473

(22) International Filing Date: 2 March 1999 (02.03.99)

(30) Priority Data:

09/033,462

2 March 1998 (02.03.98)

(71) Applicant (for all designated States except US): TRUSTEES

OF TUFTS COLLEGE [US/US]; Tufts University, Ballou
Hall, Medford, MA 02155 (US).

(72) Inventors; and

(75) Inventors/Applicants (for US only): WALT, David, R.
[US/US]; 4 Candlewick Close, Lexington, MA 02178
(US). TAYLOR, Laura [US/US]; 15A Bradbury Avenue,
Medford, MA 02155 (US).

(74) Agent: CREEHAN, R., Dennis; P.O. Box 750070, Arlington
Heights, MA 02175-0070 (US).

(81) Designated States: AL, AM, AT, AU, AZ, BA. BB, BG, BR,
BY. CA, CH. CN, CU, CZ, DE, DK, EE, ES, FL GB, GD,
GE, GH, GM, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR,
KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN,
MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK,
SL, TJ, TM, TO, TT, UA, UG, US, UZ, VN, YU, ZW,
ARIPO patent (GH, GM, KE, LS, MW, SD, SL, SZ, UG,
ZW), Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ,
TM), European patent (AT, BE, CH, CY, DE, DK, ES, FI,
FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OAPI patent
(BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE.
SN, TD, TG).

Published

Without international search report and to be republished
upon receipt of that report.

(54) Title: BIOSENSOR ARRAY COMPRISING CELL POPULATIONS CONFINED TO MICROCAVITIES
(57) Abstract

A biosensor, sensor
array, sensing method
and sensing apparatus
are provided in which
individual cells or randomly
mixed populations of cells,
having unique response
characteristics to chemical
and biological materials, are
deployed in a plurality of
discrete sites in a substrate.
In a preferred embodiment,
the discrete sites comprise
microwells formed at the
distal end of individual
fibers within a fiber optic
array. The biosensor
array utilizes an optically
interrogatable encoding
scheme for determining
the identity and location of
each type in the array and
provides for simultaneous
measurements of large
numbers of individual cell
responses to target analytes.
The sensing method utilizes
the unique ability of cell
populations to respond to

biologically* significant compounds in a characteristic and detectable manner. The biosensor array and measurement method may be
employed in the study of biologically active materials, in situ environmental monitoring, monitoring of a variety of bioprocesses, and for
high throughput screening of large combinatorial chemical libraries.

Scanning Electron Micrograph (SEM) of a single NEH 3T3 mouse fibroblast cell in an etched
microwcll. Each well h» a diameter of 7pm. a deprfi of 5pm. and a volume of - !45fL.

FOR THE PURPOSES OF INFORMATION ONLY
Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.

Albania

Spain

Lesotho

Slovenia

Armenia

Finland

Lithuania

Slovakia

Austria

France

Luxembourg

Senegal

Australia

Gabon

Latvia

Swaziland

Azerbaijan

United Kingdom

Monaco

Chad

Bosnia and Herzegovina

Georgia

Republic of Moldova

Togo

Barbados

Ghana

Madagascar

Tajikistan

Belgium

Guinea

The former Yugoslav

Turkmenistan

Burkina Faso

Greece

Republic of Macedonia

Turkey

Bulgaria

Hungary

Mali

Trinidad and Tobago

Benin

Ireland

Mongolia

Ukraine

Brazil

Israel

Mauritania

Uganda

Belarus

Iceland

Malawi

United States of America

Canada

Italy

Mexico

Uzbekistan

Central African Republic

Japan

Niger

Viet Nam

Congo

Kenya

Netherlands

Yugoslavia

Switzerland

Kyrgyzstan

Norway

Zimbabwe

C6te d'lvoire

Democratic People's

New Zealand

Cameroon

Republic of Korea

Poland

China

Republic of Korea

Portugal

Cuba

Kazakstan

Romania

Czech Republic

Saint Lucia

Russian Federation

Germany

Liechtenstein

Sudan

Denmark

Sri Lanka

Sweden

Estonia

Liberia

Singapore

WO 99/45357 PCT/US99/04473
BIOSENSOR ARRAY COMPRISING CELL POPULATIONS CONFINED TO MICROCAVITIES

FIELD OF THE INVENTION
The present invention is generally concerned with biosensors, biosensor arrays, and sensing
apparatus, and sensing methods for the analysis of chemical and biological materials. More
particularly, the invention is directed to biosensors, biosensor arrays, sensing apparatus and sensing
methods which employ cells and mixed populations of cells for analysis of chemical and biological
materials.

BACKGROUND OF THE INVENTION
It is generally recognized that important technical advances in chemistry, biology and
medicine benefit from the ability to perform microanalysis of samples in minute quantities. However,
making analytical measurements on minute quantities has long been a challenge due to difficulties
encountered with small volume sample handling, isolation of analytes, and micro-analysis of single-
cell physiology.

Nanoliter, picoliter, and femtoliter volume studies have been explored in a range- of
applications involving in vitro and in vivo cellular investigations [R.M. Wightman, et al., Proc. Natl.
Acad. Sci. U.S.A. 88:10754(1991); R.H.Chow, etal. Nature 356:60(1992); T.K. Chen, et al. Anal.
Chem. 66:3031(1994); S.E. Zerby, et al., Neurochem. 66:651(1996); P.A. Garis, et al. J.Neurosci.
14:6084(1994); G.Chen, et al., J.Neurosci. 15:7747(1995)], electrochemistry [R.A. Clark, et al., Anal.
Chem. 69(2):259(1997)], matrix-assisted laser desorption- ionization mass spectrometry [S.
Jespersen, et al., Rapid Commun. Mass Spectrom. 8:581(1994)], micro-column liquid
chromatography [I.A.Holland, etal., Anal.Chem. 67:3275(1995); M.D. Oates, et al., Anal. Chem.
62:1573(1990)], micro-titration [M. Gratzl, et al Anal.Chem. 65:2085(1993); C.Yi, et al.. Anal.Chem.
66:1976(1994)], and capillary electrophoresis [M.Jansson, et al., J.Chromatogr. 626:310(1992);
P.Beyer Hietpas, et al. J.Uq.Chromatogr. 18:3557(1995)].

Clark, et al. [Anal.Chem. 69(2):259(1997)] has disclosed a method for fabricating picoliter
microvials for electrochemical microanalysis using conventional photolithographic masking and
photoresist techniques to transfer mold polystyrene microvials on silicon wafer templates. These

WO 99/45357 PCT/US99/04473

microvials typically exhibit non-uniformity in size and shape due to the difficulty in controlling the resist
etching of the molding surface and the transfer molding process.

Park, et al. [Science 276:1401(1997)] has disclosed a modified lithographic method for
producing arrays of nanometer-sized holes using polystyrene-polybutadiene, ordered, diblock
5 copolymers as masks in reactive ion etching of silicon nitride. This multi-step method is capable of
producing arrays of picoliter-sized holes which are typically 20 nanometers in diameter and 20
nanometers deep with a spacing of 40 nanometers. Hole densities of up to 1 0 1 1 holes/cm 2 are
disclosed. The range of sizes and spacings of the holes produced by this method is limited by the size
of the copolymer microdomains. Uniformity of hole size and spacing is difficult to maintain with this

10 method due to difficulties in controlling the etching method employed to form the holes.

Deutsch, et al. [Cytometry 16:214(1994)] have disclosed a porous electroplated nickel
microarray comprised of micron-sized conical holes in blackened nickel plate. Hole sizes range from a
7 urn upper diameter to a 3 urn lower diameter with an 8 urn depth. The array is used as a cell carrier
for trapping individual cells while studying the responses of individual cells to changes in their

15 microenvironment. In U.S. Patent 4,772540, Deutsch, et al., have also disclosed a method for making
such an array using a combined photoresist and electroplating technique.

Corning Costar Corp. (Acton, Ma) produces a commercial microwell array for miniaturized
assays under the trademark PixWell™. These arrays are made from microformed glass plates and
comprise 40 urn diameter by 20 urn deep tapered wells with a well density of 4356 wells/cm 2 .

2 0 Microwell arrays have particular utility in the study of living cells. In cell research, the

measurement of responses of individual cells to changes or manipulations in their local environment is
desirable. Any method or device designed for such studies must provide for the capability of
maintaining cell viability, identifying the location of individual cells, and correlating response
measurements with individual cells.

2 5 Due to the availability of viable fluorescent probes for intracellular studies, fluorescence

measurements of living cells have significant utility in the study of cell functions. Thus fluorescence
optical measurements are often utilized in cell studies where three generic methods of cell
measurement are available, comprising bulk measurements of cell populations, dynamic
measurements of cell populations or individual cells, and static measurements of individual cells.

WO 99/45357 PCT/US99/04473
The characteristics of an entire cell population as a whole can be studied with bulk
measurements of sample volumes having a plurality of cells. This method is preferred where cell
populations are very homogeneous. A generally recognized limitation of this method is the presence
of background fluorescence which reduces the sensitivity of measurements and the inability of
5 distinguishing differences or heterogeneity within a cell population.

Flow cytometry methods are often employed to reduce problems with background
fluorescence which are encountered in bulk cell population measurements [M.R.Gauci, et al.,
Cytometry 25:388(1996); R.C.Boltz, et a!., Cytometry 17:128(1994)]. In these methods, cell
fluorescence emission is measured as cells are transported through an excitation light beam by a
10 laminar flowing fluid. Flow cytometry methods may be combined with static methods for preliminary
sorting and depositing of a small number of cells on a substrate for subsequent static cell
measurements [U.S. Patent No. 4,009,435 to Hogg, et al.; Kanz, et al., Cytometry 7:491 (1986);
Schildkraut, et al., J.Histochem Cytochem 27,289(1979)].

Gauci, et al,, disclose a method where cell size, shape and volume is measured by light
15 scattering and fluorescent dyes are utilized to determine protein content and total nucleic acid content
of cells. This method further provides for counting and sizing various cells at a rate of approximately
100 cells per second.

Flow cytometry techniques are generally limited to short duration, single measurements of
individual cells. Repetitive measurements on the same cell over time are not possible with this
2 0 method since typical dwell times of a cell in the excitation light beam are typically a few microseconds.
In addition, the low cumulative intensity from individual cell fluorescence emissions during such short
measurement times reduces the precision and limits the reliability of such measurements.

Regnier, et al., [Trends in Anai.Chem. 14(4):1 77(1 995)] discloses an invasive,
electrophoretically mediated, microanalysis method for single cell analysis. The method utilizes a

2 5 tapered microinjector at the injection end of a capillary electrophoresis column to pierce an individual

cell membrane and withdraw a sample of cytoplasm. The method measures cell contents, one cell at
a time. The method is generally limited to the detection of easily oxidized species.

Hogan, et al., [Trends in AnaLChem. 12(1):4(1993)] discloses a microcolumn separation
technique which may be utilized in combination with either a conventional gas chromatograph-mass

3 0 spectrometer, micro thin layer chromatography or high pressure liquid manipulation of small cellular

WO 99/45357 PCT/US99/04473

volumes. The sensitivity of the method is limited and may require pre-seiection of target compounds
for detection.

Static methods are generally the preferred method for measurements on individual cells.
Measurement methods range from observing individual cells with a conventional optical microscope to
5 employing laser scanning microscopes with computerized image analysis systems [see L. Hart, et al. (
Anal Quant Cytol. Histol. 12:127(1990)]. Such methods typically require the attachment of individual
cells to a substrate prior to actual measurements. Problems are typically encountered in attaching
single cells or single layers of cells to substrates and in maintaining cells in a fixed location during
analysis or manipulation of the cell microenvironment. Additionally, repetitive measurements on

10 individual cells typically require physically indexing the location of individual cells and providing a
mechanism for scanning each cell sequentially and returning to indexed cell locations for repeated
analysis of individual cells.

Huang, et al., [Trends in Anal Chem., 14(4)158(1995)] discloses a static electrochemical
method and electrode for monitoring the biochemical environment of single cells. The method

15 requires fabrication and manual positioning of a microelectrode reference and working electrode
within the cell. The method has been used to detect insulin, nitric oxide and glucose inside single
cells or external to the cells. The method is generally limited to the study of redox reactions within
cells.

Ince, et al. [J.Immunol Methods 128:227(1990)] disclose a closed chamber device for the
2 0 study of single cells under controlled environments. This method employs a micro-perfusion chamber
which is capable of creating extreme environmental conditions for cell studies. Individual cells are
held in place by two glass coverslips as various solutions are passed through the chamber. One
limitation of the method is the difficulty in eliminating entrapped gas bubbles which cause a high
degree of autofluorescence and thus reduces the sensitivity of measurements due to background

2 5 fluorescence.

In an attempt to overcome the limitations encountered with conventional static methods,
Deutsch, et al., [Cytometry 16:214(1994)] and Weinreb and Deutsch, in U.S. Patent Nos. 4,729,949,
5,272,081 , 5,310,674, and 5,506,141 , have disclosed an apparatus and method for repetitive optical
measurements of individual cells within a cell population where the location of each cell is preserved

3 0 during manipulation of the cell microenvironment.

WO 99/45357 PCT/US99/04473
A central feature of the apparatus disclosed by Deutsch, et al., is a cell carrier, comprising a
two dimensional array of apertures or traps which are conical-shaped in order to trap and hold
individual cells by applying suction. The cell carrier is typically fabricated by the combined
electroplating-photoresist method disclosed in U.S. Patent 4,772540 to Deutsch, et al. The purpose
5 of the cell carrier is to provide a means for maintaining the cells in fixed array locations while

manipulating the cell environment. Individual cells are urged into cell carrier holes by suction and the
wells are subsequently illuminated with a low-intensity beam of polarized light that reads back-emitted
polarization and intensity. Measurements are compared when two different reagents are sequentially
reacted with the ceils. The method as disclosed requires two separate cell carriers for both a baseline

10 control and analyte measurement.

The method and device of Deutsch, et al., have been employed by pathologists in diagnostic
tests to determine the health and viability of cell samples taken from patients. The method and device
have been applied to both cancer screening [Deutsch, et al., Cytometry 16:214(1994), Cytometry
23:159(1996), and European J. Cancer 32A(10): 1758(1 996)] and rheumatoid arthritis [Zurgil, et al.,

15 isrJ.Med.Sci. 33:273(1997)] in which fluorescence polarization measurements are used to

differentiate lymphocytes of malignant versus healthy cells based on changes in the internal viscosity
and structuredness of the cytoplasmic matrix induced by exposure to tumor antigen and mitogens.

The method and device disclosed by Deutsch, et al., requires employment of a scanning table
driven by three stepping motors and a computer control system for mapping, indexing and locating

2 0 individual cells in the cell carrier. The use of such mechanical scanning methods introduces

limitations in reproducibility and accuracy of measurements due to conventional mechanical problems
encountered with backlash and reproducible positioning of individual cell locations for repeated
measurements. In addition, mechanical scanning of the entire array prolongs the measurement time
for each cell in the array.

2 5 The method disclosed by Deutsch, et al., is further limited by the use of fluorescence

polarization measurements which have certain intrinsic limitations due to the significant influence of
various optical system components on polarization as the fluorescence emission response is passed
from the cell carrier to optical detectors. Birindelli, et al. [European J. Cancer 33(8): 1333(1 997)], has
also identified limitations in this method due to fluctuations in electropolarisation values which require

3 0 taking averages of at least three measurement scans for each condition so as to obtain reliable

WO 99/45357 PCT/US99/04473

measurements. In addition, for cell studies, polarization measurements are generally limited to celt
responses which produce sufficient changes in cytoplasm viscosity to produce a detectable change in
polarization. Since not all cell responses are accompanied by detectable viscosity changes, the
method is further limited to the cell activities which create such viscosity changes in the cytoplasm.
5 Zare, et a!., [Science 267:74(1995); Biophotonics International, March-April, p17 (1995)]

discloses a biosensor system based on the response of living cells to complex biological materials
fractionated by a microcolumn separation technique. Cells which were positioned on a glass cover
slip were treated with a fluorescent probe and subsequently shown to be sensitive to a series of
biological compounds including acetylcholine, bradykinin, and adenosine triphosphate as well as

10 changes in intracellular calcium levels.

Yeung, et al. [Acc. Chem. Res. 27:409(1994)] has reviewed a number of methods for single
cell response studies and has observed a significant variation and heterogeneity within cell
populations based on analyte measurements. For example, the reference discloses a capillary
electrophoresis method for exposing cells to biologically reactive compounds, extracting the

15 intracellular fluid of individual cells produced in response to such compounds, and identifying analytes
from migration times in the capillary column. Other fluorescence-based assays are also disclosed.
Significant cell-to-cell variations and heterogeneity in individual cell responses within a cell population
were observed which differences could provide a means for discriminating between biological and
chemical compounds in contact with individual cells.

20 McConnell, et al. [Science, 257:1906(1992)], disclose a microphysiometer device known as

the "Cytosensor" which uses a light addressable potentiometer sensor to measure the rate at which
cells acidify their environment. This sensor acts as miniaturized pH electrode for monitoring cell
responses which produce detectable changes in local pH. The disclosed device is limited to the
measurement of proton excretions from cells and thus is only capable of detecting acidic cell

2 5 responses to analytes.

U.S. Patent No. 5,177,012 to Kim, et al., disclose a biosensor for the determination of glucose
and fructose. The biosensor is produced by treating whole cells with an organic solvent and
immobilizing the treated cells residue on a support to form a whole cell membrane which is applied to
a pH electrode.

WO 99/45357 PCT/US99/04473

U.S. Patent No. 5,690,894 to Pinkel, et al., discloses a biosensor which employs biological
"binding partners", materials such as nucleic acids, antibodies, proteins, lectins and other materials
derived from cells, tissues, natural or genetically-engineered organisms. These agents are used in
conjunction with a fiber optic array where each species of binding partners is uniquely addressed by
5 a group of fibers within the fiber optic bundle which is coupled to an optical detector. The array was
designed for screening of extensive arrays of biological binding partners.

While many of the prior art methods provide for the analysis of either single cells or
populations of cells and some of these methods provide for monitoring cell responses to target
analytes, none of the disclosed methods provides for employing large populations of monocultures or

1 0 mixed populations of living cells for simultaneously monitoring the responses of individual cells to

biological stimuli produced by chemical and biological analytes. Thus there is a need for a biosensor
array and method which efficiently utilizes the ability of populations of living cells to respond to
biologically significant compounds in a unique and detectable manner. Since the selectivity of living
cells for such compounds has considerable value and utility in drug screening and analysis of

15 complex biological fluids, a biosensor which makes use of the unique characteristics of living cell
populations would offer distinct advantages in high throughput screening of combinatorial libraries
where hundreds of thousands of candidate pharmaceutical compounds must be evaluated. In
addition, such a sensor would be useful in monitoring bioprocesses and environmental pollution
where the enhanced sensitivity of living cells to their environment can be exploited.

SUMMARY OF THE INVENTION
In general, the invention provides for a biosensor, a biosensor array, a biosensor sensing
system and sensing methods for the analysis of chemical and biological materials. More particularly,
the invention provides for biosensors and biosensor arrays, sensing apparatus and sensing methods

2 5 which employ living cells and mixed populations of living cells for analysis of chemical and biological

materials.

The biosensor array of the present invention comprises either a monoculture of living cells or
randomly mixed populations of living cells wherein each individual cell in the array is positioned on a
substrate at an optically-addressable, discrete site which accomodates the size and shape of

3 0 individual cells. In one embodiment, the discrete site comprises a microwell or microcavity which is

WO 99/45357 PCT/US99/04473

preformed to accommodate the size and shape of the individual cells. The biosensor array sensing
method relies on the well known fact that individual ceils, which are chemically or biologically
stimulated by the presence of a biological or chemical material in the cell environment, will respond by
producing a change in the cell or cellular environment which can be optically interrogated and
5 detected within the cell itself or from an indicator compound, for example, a fluorophore, chromophore
or dye, either attached to the ceil, taken up in the cell, or added to the local cell environment. The
biosensor of the present invention thus capitalizes on the ability of living cells to respond to
biologically significant compounds. Since the selectivity of living cells for such compounds has
considerable value and utility in drug screening and analysis of complex biological fluids, the

1 0 biosensor of the present invention offers distinct advantages to high throughput screening of

combinatorial libraries where hundreds of thousands of candidate compounds must be evaluated.

As will be appreciated by one skilled in the art, a variety of substrate materials and substrate
configurations may be employed with the biosensor array of the present invention. Any substrate or
material that can be adapted to provide discrete sites that are appropriate for the attachment or

1 5 association of individual cells and is accessible for optical interrogation of the cell array and detection
of cell responses would be particularly useful as an array substrate. Candidate substrate materials
include, but are not limited to, glasses, polymers, ceramics, metals, and composites formed from
these materials. Substrate surface geometries may be planer, spherical, concave, convex, and
textured. Preferably, a suitable substrate would be configured so as to provide for optical

2 0 interrogation of the array and detection of cell responses to analytes of interest.

In a preferred embodiment, the biosensor array of the present invention is incorporated into a
fiber optic array which serves as a substrate for cell placement. By "fiber optic array" or other
grammatical equivalents herein is meant a plurality of individual fibers or fiber strands that may be
either grouped together as discrete, individual fibers in a fiber bundle or, alternatively, joined along
25 their axial dimensions as a preformed unitary fiber optic array. The individual fibers in such an array
may be arranged in a uniform or coherent configuration, for example as in an imaging fiber, or may be
randomly oriented and incoherent. When a fiber optic array is employed as a sensor substrate, the
distal end of each fiber in a fiber optic bundle or fiber optic array is chemically etched so as to create a
cavity or microwell. A schematic diagram of the biosensor array concept of the present invention is

3 0 shown in Fig. 1 . In a preferred embodiment, individual living cells of either a monoculture of cells or

WO 99/45357 PCT/US99/04473

mixed populations of cell lines are deployed in the microwells. The microwells are formed by
anisotropic etching of the cores of the individual fiber in the fiber bundle or fiber array. The microwells
are formed by controlling the etching process so as to remove a centralized core portion of the
individual fiber strands while leaving the surrounding cladding intact. The resultant etched cavity is
5 dimensioned for accommodating an individual cell. By selecting a fiber optic bundle or fiber optic
array whose individual fiber cores are appropriately sized and by careful control of the etching
conditions, the diameter and depth of the microwells can be controlled and adjusted over any
convenient dimension range so as to match the size of any desired cell type.

In one embodiment, either discrete substrate cites or the interior surfaces of the microwells
10 may be coated with a thin film of biologically compatible material such as collagen, fibronectin,

polylysine, polyethylene glycol, polystyrene, or a metal such as gold, platinum or palladium. In an
alternative embodiment, an indicator compound, for example, a fluorophore, a chromophore or dye,
may be attached to the microwell surface for detecting cell responses to chemical or biological
stimulation.

15 By incorporating a biosensor into an optically interrogatable substrate or a fiber optic array,

the innovation of the biosensor of the present invention is in providing for optical coupling of individual
cells located at discrete substrate cites or microwells with discrete detector elements, CCD cameras,
or individual optical fibers in a fiber optic array or bundle that are in optical communication with such
devices. By "optical coupling", "optical communication", or "optical cooperation" or other grammatical

2 0 equivalents herein is meant the capability of either optically stimulating individual cells within the

biosensor array with excitation light or optically interrogating the optical response of individual cells
within the array to analytes, by conveying light to and from individual cells located at discrete cites
within the array using either conventional optical train elements or optical fibers. Since typical fiber
optic arrays contain thousands of discrete individual fiber strands, the invention thus provides for the

2 5 individual optical coupling and interrogation of thousands of cells within an array, thereby providing for
a large number of independent cell response measurements for each cell population within an array.
Due to both the number of cell populations available and the correspondingly large number of
individual cells within each cell population, a significant innovation of the present invention is in
providing for the summing and amplification of the characteristic optical response signatures of

WO 99/45357 PCT/US99/04473

multiple independent measurements taken from cells within each cell population, thereby improving
the detection limit and sensitivity of the biosensor.

An additional innovation of the present invention is that, by deploying a large number of cell
populations within the array, and providing a large number of individual cells in each population, the
5 discriminating capabilities of the biosensor array toward biological or chemical analytes is significantly
enhanced by providing for thousands of cell responses from a large number of cell populations. This
feature directly mimics the actual behavior of the human olfactory system where the combined signals
from thousands of receptor cells, in each grouping of nearly a thousand different receptor cell types
found in the epithelium layer, none of which are particularly sensitive in themselves, lead to a highly

10 amplified sensory response to odors [see Kauer, et al, Trends Neurosci. 14:79(1991). One

embodiment of the present invention thus mimics the evolutionary scent amplification process found
in the human olfactory system in order to significantly enhance biosensor array sensitivity to analytes
by summing the low-level responses of a large number of cells in the biosensor array. By summing
the responses from a number of cells at low analyte concentrations, a substantial improvement in

15 signal-to-noise ratio can be achieved and a corresponding reduction in the detection limit of the
biosensor array is obtained.

A unique feature of the biosensor array of the present invention is that each of the individual
cells and cell populations in the array may be encoded for maintaining cell type identity and location
where randomly mixed populations of cells are employed. Cells may be encoded prior to disposing

2 0 them in the microwells or, alternatively, following placement in the microwells. The invention provides
for either encoding randomly mixed individual cells and cell populations with a fluorophoric or
chromophoric dye compound or, alternatively, using self-encoded cells which are either naturally
fluorescing or genetically engineered to fluoresce. Although cell populations may be randomly mixed
together, this innovative feature provides for the identity and location of each cell type to be

2 5 determined via a characteristic optical response signature when the eel! array is either illuminated by

excitation light energy or, alternatively, subjected to biological stimuli.

In one embodiment, cells or cell populations may be self-encoded by selecting cell
populations, such as green fluorescent protein mutants, which exhibit either chemiluminescence,
bioluminescence, or whose optical response to biological stimuli yield a unique detectable

3 0 fluorescence signal. Other cell populations may be employed where cells within a population yield a

WO 99/45357 PCT/US99/04473

unique temporal optical response to stimuli. Either naturally occurring of genetically engineered cell
lines may be utilized.

In various alternative embodiments, cells may be encoded with dye compounds which are
attached to cells, taken up by cells or provided in the local cell environment. Examples of useful
5 encoding dyes include fluorophores, chromophores, stains or a dye compounds. For example,
conventional cell fluorophore probes such as fluoresceins, rhodamines, naphthalimides,
phycobiliproteins, nitrobenzoxadiazole may be utilized. A particularly useful reference for selecting
appropriate encoding dyes is R.P. Haugland, Handbook of Fluorescent Probes and Research
Chemicals (6 th ed.), Molecular Probes lnc.(Eugene, OR, 1996) which is herein incorporated by this
1 0 reference.

When dye compounds are employed for encoding, cell populations within the biosensor array
may be readily decoded by exciting the array with excitation light and indexing cells types within
randomly dispersed populations by their response to excitation. In one embodiment, a single
fiuorophoric or chromophoric material or dye is used for encoding the cells. In an alternative

15 embodiment, two or more encoding materials or dyes may be used to encode cell populations and the
optical response intensity ratios for the dyes, produced by exposure to excitation light energy, are
employed to encode and identify members of the cell population with the array. In an alternative
embodiment, cells may be decoded by excitation light when exposed to a common analyte. In
another embodiment, encoded cells may be decoded by their response to a generic cell activator

2 0 using either a pH or Ca +2 indicator.

The innovative cell encoding feature of the present invention overcomes certain limitations of
prior art devices by eliminating the need for mechanically scanning the array, mechanically indexing
the location of cells, and mechanically positioning the array for measurements of individual celts within
the array. The invention thus provides for rapid, simultaneous measurements of all cells and cell

2 5 populations within the array without the need to mechanically scan the array to acquire a series of

sequential measurements for each cell. Thus monitoring and measuring the responses of all cells in
the array occurs simultaneously without a prolonged delay between the first cell measurement and
last cell measurement. The ability to measure all cell responses simultaneously thus provides for the
capability to monitor both short term cell response and long term cell response. This innovative

3 0 feature thus enables the monitoring of rapid biologically significant cell processes and cell responses

WO 99/45357 PCT/US99/04473

on a short time scale. In addition, the ability to simultaneously measure cell responses over a short
time scale enables the measurement of individual cell and cell population response rates to changes
in the biosensor array environment. This feature thus provides for additional discriminating response
information which is useful for detecting biological or chemical analytes.
5 The biosensor array of the present invention can employ either naturally occurring cells and

celi populations or genetically engineered cell lines. Virtually any cell type and size can be
accommodated by matching the cell size to individual optical fiber optic core diameters and etching
conditions. In one embodiment, NIH 3T3 mouse fibroblast cells were employed. In alternative
embodiments, other cells types such as e. coli bacteria, staphylococcus bacteria, myoblast precursors

10 to skeletal muscle cells, neutrophil white blood cells, lymphocyte white blood cells, erythroblast red
blood cells, osteoblast bone cells, chondrocyte cartilage cells, basophil white blood cells, eosinophil
white blood cells, adipocyte fat cells, invertebrate neurons (Helix aspera), mammalian neurons, or
adrenomedullary cetls, may be utilized as well. Any cell type or mixtures of cell population types may
also be employed providing the microwell can accommodate the individual cell size.

15 The optical responses of individual cells and cell populations to chemical or biological stimuli

are typically interrogated and detected by coupling individual cells with appropriate indicators which
may be either fluorophores, chromophores, stains or a dye compounds. For example, conventional
cell fluorophore probes such as fluoresceins, rhodamines, naphthalimides, phycobiliproteins,
nitrobenzoxadiazole may be utilized. Alternatively, permeant or impermeant cell membrane potential

2 0 indicators, ion indicators, reactive oxygen indicators and pH indicators may be employed. A

particularly useful reference for selecting appropriate indicators is R.P. Haugland, Handbook of
Fluorescent Probes and Research Chemicals (6 th ed.), Molecular Probes lnc.(Eugene, OR, 1996)
which is herein incorporated by this reference. Any suitable indicator or combinations of indicators
may be utilized provided the indicator does not compromise cell response. In a variety of alternative
25 embodiments, indicators may be either incorporated directly into the cell, for example by attachment
to the cell membrane, by absorption or injection into the cell cytoplasm, or added to the cell external
environment, such as a fluid contained within the microwells. In an alternative embodiment, indicators
may be attached to the surface of the microwells.

Individual cells or arrays of cells and cell populations may be optically interrogated and cell

3 0 responses to analytes may be measured by conventional optical methods and instrumentation that

WO 99/45357 PCT/US99/04473

are known to those skilled in the art. Cells may be optically interrogated with any suitable excitation
light energy source, such as arc lamps, lasers, or light emitting diodes, that are capable of producing
light at an appropriate wavelength for exciting dye indicators that may be employed for encoding cell
populations or for responding to analytes of interest. The optical responses of individual cells or cell
5 populations may be monitored and measured with any suitable optical detection means, including, but
not limited to film or conventional optical detectors, such as photoresistors, photomultiplier tubes,
photodiodes, or charge coupled device (CCD) cameras. In a preferred embodiment, CCD cameras
are employed to capture fluorescent images of the biosensor array for detecting responses of each
cell and various cell subpopulations to analytes. In this embodiment, both individual cell responses

1 0 and a captured image of the array response may be employed for detecting analytes.

In summary, the biosensor array and sensing method of the present invention offers many
distinct advantages in overcoming the limitations of prior art devices. The sensor arrays are easily
fabricated from commercially available optical imaging fibers to yield a cost effective, high density,
precisely formed, biosensor array without requiring any sophisticated machining or forming process.

15 Since optical fibers and fiber optic arrays are available in a wide variety of fiber core diameters, most
cell types and sizes may be accommodated in by the device and method of the present invention. In
addition, cells can be readily dispersed into the microwell array in random fashion with no need for
physical indexing or scanning to locate individual cells or cell populations due to the innovative cell
encoding technique. Sensing methods and sensing systems which employ the biosensor and sensor

2 0 array of the present invention avoids many of the limitations in manipulating cells encountered with

prior art devices. Once cells are placed within the microwells of the array, conventional imaging
systems and methods which employ an imaging camera and conventional optics, can monitor the
response of thousands of cells simultaneously, eliminating requirements for mechanical scanning
mechanisms. Analysis of measurement data is further facilitated by implementing commercially
25 available imaging software to process images of the biosensor array using pattern recognition
techniques combined with neural network and other statistical methods.

The biosensor array and sensing method of the present invention may be employed for a
number of useful analytical applications where individual cells, which are chemically or biologically
stimulated by the presence of a biological or chemical material in the local cell environment, will

3 0 respond to their environment by producing an optically detectable response either due to the

WO 99/45357 PCT/US99/04473

presence of an appropriate indicator or due to the characteristic optical response of particular cell
types which exhibit either natural or genetically-engineered chemiluminescence or bioluminescence.
The biosensor array and method of the present invention thus capitalizes on the ability of living cells
to respond to biologically significant compounds. Since the selectivity of living cells for such
5 compounds has considerable value and utility in drug screening and analysis of complex biological
fluids, the biosensor of the present invention offers distinct advantages to high throughput screening
of combinatorial libraries where hundreds of thousands of candidate compounds must be evaluated.

The above and other features of the invention, including various novel details of construction
and methods, and other advantages, will now be more particularly described with reference to the
1 0 accompanying drawings and claims. It will be understood to one skilled in the art that the particular
apparatus and method embodying the invention are shown by way of illustration and not as a
limitation of the invention. The principles and features of the invention may be employed in various
and numerous embodiments without departing from the scope of the invention.

15 BRIEF DESCRIPTION OF THE DRAWINGS

The invention is pointed out with particularity in the appended claims. Other features and
benefits of the invention can be more clearly understood with reference to the specification and the
accompanying drawings in which:

Fig. 1 is a schematic diagram of the biosensor array concept of the present invention;
2 0 Figs. 2a-b are atomic force microscope photomicrographs of a microwell array used in a

biosensor array of the present invention;

Fig. 3 is a schematic diagram of the method for depositing cells in microwells used for a
biosensor array of the present invention;

Fig. 4 is an SEM photomicrograph of a single NIH 3T3 mouse fibroblast cell in a microwell;

2 5 Fig. 5 is a schematic representation of a method for establishing cell viability of cell

populations within the biosensor array of the present invention;

Fig. 6 is a characteristic fluorescence image pattern identifying the location of a cells which
test positively for cell viability in a biosensor array of the present invention;

Fig. 7 shows the temporal response of the combined fluorescence intensity of the viable cell

3 0 population identified in Fig. 6;

WO 99/45357 PCT/US99/04473

Fig. 8 is a fluorescence optical image of an encoded cell population within a biosensor array
of the present invention;

Fig. 9 is a schematic block diagram of the measurement system used for optical
measurements of the microwell sensor array;
5 Fig. 10 is a plot of average BCECF fluorescence for nine biosensor array cells over time; and

Figs. 1 1a-b compares the viability of cells inserted in microwells both with and without a
pretreatment for filling the microwells with culture media.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

10 A. Fabrication of Microwell Arrays

The present invention provides array compositions comprising at least a first substrate with a
surface comprising individual sites. By "array" herein is meant a plurality of cells in an array format;
the size of the array will depend on the composition and end use of the array. Arrays containing from
about 2 different cells to many millions can be made, with very large fiber optic arrays being possible.

1 5 Generally, the array will comprise from two to as many as a billion or more, depending on the size of
the cells and the substrate, as well as the end use of the array, thus very high density, high density,
moderate density, low density and very low density arrays may be made. Preferred ranges for very
high density arrays are from about 10,000,000 to about 2,000,000,000 (all numbers herein are per
cm2), with from about 100,000.000 to about 1 ,000,000,000 being preferred. High density arrays

2 0 range about 1 00,000 to about 10,000,000, with from about 1 ,000,000 to about 5,000,000 being
particularly preferred. Moderate density arrays range from about 10,000 to about 100,000 being
particularly preferred, and from about 20,000 to about 50,000 being especially preferred. Low density
arrays are generally less than 10,000, with from about 1 ,000 to about 5,000 being preferred. Very low
density arrays are less than 1,000, with from about 10 to about 1000 being preferred, and from about

2 5 100 to about 500 being particularly preferred. In some embodiments, the compositions of the

invention may not be in array format; that is, for some embodiments, compositions comprising a single
cell may be made as well. In addition, in some arrays, multiple substrates may be used, either of
different or identical compositions. Thus for example, large arrays may comprise a plurality of smaller
substrates.

WO 99/45357 PCT/US99/04473

In addition, one advantage of the present compositions is that particularly through the use of
fiber optic technology, extremely high density arrays can be made. Thus for example, because cells
frequently may be 200 fm or less and very small fibers are known, it is possible to have as many as
250,000 or more (in some instances, 1 million) different fibers and cells in a 1 mm2 fiber optic bundle,
5 with densities of greater than 15,000,000 individual cells and fibers (again, in some instances as
many as 25-50 million) per 0.5 cm2 obtainable.

By "substrate" or "solid support" or other grammatical equivalents herein is meant any
material that can be modified to contain discrete individual sites appropriate for the attachment or
association of cells and is amenable to at least one detection method. As will be appreciated by
1 0 those in the art, the number of possible substrates is very large. Possible substrates include, but are
not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and
copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes,
TeflonJ, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials
including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, optical fiber bundles,
15 and a variety of other polymers. In general, the substrates allow optical detection and do not
themselves appreciably fluorescese.

Generally the substrate is flat (planar), although as will be appreciated by those in the art,
other configurations of substrates may be used as well; for example, three dimensional configurations
can be used, for example by embedding the cells in a porous block of plastic that allows sample

2 0 access to the cells and using a confocal microscope for detection. Similarly, the cells may be placed

on the inside surface of a tube, for flow-through sample analysis to minimize sample volume.
Preferred substrates include optical fiber bundles as discussed below, and flat planar substrates such
as glass, polystyrene and other plastics and acrylics.

In a preferred embodiment, the substrate is an optical fiber bundle or array, as is generally
25 described in U.S.S.N.s 08/944,850 and 08/519,062, PCT US98/05025, and PCT US98/09163, all of
which are expressly incorporated herein by reference. Preferred embodiments utilize preformed
unitary fiber optic arrays. By "preformed unitary fiber optic array" herein is meant an array of discrete
individual fiber optic strands that are co-axially disposed and joined along their lengths. The fiber
strands are generally individually clad. However, one thing that distinguished a preformed unitary

3 0 array from other fiber optic formats is that the fibers are not individually physically manipulatable; that

WO 99/45357 PCT/US99/04473

is, one strand generally cannot be physically separated at any point along its length from another fiber
strand. In general, the discussion herein is focused on fiber optic arrays, although as will be
appreciated by those in the art, other substrates as described above may be used in any embodiment
described herein.

5 At least one surface of the substrate is modified to contain discrete, individual sites for later

association of cells. These sites may comprise physically altered sites, i.e. physical configurations
such as wells or small depressions in the substrate that can retain the cells, such that a cell can rest
in the well, or the use of other forces (magnetic or compressive), or chemically altered or active sites,
such as biologically or chemically functionalized sites, electrostatically altered sites, hydrophobically/

1 0 hydrophilically functionalized sites, spots of adhesive, etc.

The sites may be a pattern, i.e. a regular design or configuration, or randomly distributed. A
preferred embodiment utilizes a regular pattern of sites such that the sites may be addressed in the X-
Y coordinate plane. "Pattern" in this sense includes a repeating unit cell, preferably one that allows a
high density of cells on the substrate. However, it should be noted that these sites may not be

1 5 discrete sites. That is, it is possible to use a uniform surface of adhesive or chemical functionalities,
for example, that allows the attachment of cells at any position. That is, the surface of the substrate is
modified to allow attachment of the cells at individual sites, whether or not those sites are contiguous
or non-contiguous with other sites. Thus, the surface of the substrate may be modified such that
discrete sites are formed that can only have a single associated cell, or alternatively, the surface of

2 0 the substrate is modified and cells may go down anywhere, but they end up at discrete sites.

In a preferred embodiment, the surface of the substrate is modified to contain microwells, i.e.
depressions in the surface of the substrate. This may be done as is generally known in the art using
a variety of techniques, including, but not limited to, photolithography, stamping techniques, pressing,
casting, molding, microetching, electrolytic deposition, chemical or physical vapor deposition

2 5 employing masks or templates, electrochemical machining, laser machining or ablation, electron

beam machining or ablation, and conventional machining. As will be appreciated by those in the art,
the technique used will depend on the composition and shape of the substrate.

In a preferred embodiment, physical alterations are made in a surface of the substrate to
produce the sites. In a preferred embodiment, the substrate is a fiber optic bundle and the surface of

30 the substrate is a terminal end of the fiber bundle, as is generally described in 08/818,199 and

WO 99/45357 PCT/US99/04473

09/151,877, both of which are hereby expressly incorporated by reference. In this embodiment, wells
are made in a terminal or distal end of a fiber optic bundle comprising individual fibers. In this
embodiment, the cores of the individual fibers are etched, with respect to the cladding, such that small
wells or depressions are formed at one end of the fibers. The required depth of the wells will depend
on the size of the cells to be added to the wells.

Generally in this embodiment, the cells are non-covalently associated in the wells, although
the wells may additionally be biologically or chemically functional ized as is generally described below,
cross-linking agents may be used, or a physical barrier may be used, i.e. a film or membrane over the
cells.

In a preferred embodiment, the surface of the substrate is modified to contain biologically or
chemically modified sites, that can be used to attach, either covalently or non-covalently, the cells of
the invention to the discrete sites or locations on the substrate. "Chemically modified sites" in this
context includes, but is not limited to, the addition of a pattern of chemical functional groups including
amino groups, carboxy groups, oxo groups and thiol groups, that can be used to attach cells which
generally also contain corresponding reactive functional groups on their surfaces; the addition of a
pattern of adhesive that can be used to bind the cells, (either by prior chemical functional ization for
the addition of the adhesive or direct addition of the adhesive); the addition of a pattern of charged
groups (similar to the chemical functionalities) for the electrostatic attachment of the cells, i.e. when
the cells comprise charged groups opposite to the sites; the addition of a pattern of chemical
functional groups that renders the sites differentially hydrophobic or hydrophiiic, such that the addition
of similarly hydrophobic or hydrophiiic cells under suitable experimental conditions will result in
association of the cells to the sites on the basis of hydroaffinity. Alternatively, biological modifications
include the use of binding ligands or binding partner pairs, including, but not limited to,
antigen/antibody pairs, enzyme/substrate or inhibitor pairs, receptor-ligand pairs, carbohydrates and
their binding partners (lectins, etc.).

In a preferred embodiment, an array of micrometer-sized wells is created at the distal face of
an optical imaging fiber by a selective etching process which takes advantage of the difference in etch
rates between core and cladding materials. This process has been previously disclosed by Pantano,
et al., Chem. Mater. 8:2832 (1996), and Walt, et al., in U.S. Patent Application 08/818,199. The etch

WO 99/45357 PCT/US99/04473

reaction time and conditions are adjusted to achieve desired control over the resultant microwell size
and volume. Microwells are thus sized to accommodate a single cell of any desired cell type.

The sensor array design can accommodate a variety of cell sizes and configurations utilizing
either commercially available optical fibers and fiber optic arrays or custom made fibers or fiber
5 arrays. The major requirement in selecting candidate fibers or fiber optic arrays for fabricating
sensors is that the individual fibers have etchable cores.

In one embodiment, the interior surfaces of the microwells may be coated with a thin film or
passivation layer of biologically compatible material, similar to the biological modifications of the
substrate as outlined above. For example, materials known to support cell growth or adhesion may
10 be used, including, but not limited to, fibronectin, any number of known polymers inclduing collagen,

polylysine and other polyamino acids, polyethylene glycol and polystyrene, growth factors, hormones,
cytokines, etc. Similarly, binding ligands as outlined above may be coated onto the surface of the
wells. In addition, coatings or films of metals such as a metal such as gold, platinum or palladium may
be employed. In an alternative embodiment, an indicator compound, for example, a fluorophore, a
1 5 chromophore or dye, may be attached to the microwell surface for detecting cell responses to
chemical or biological stimulation.

The method of fabricating microwells can be adapted to any fiber size so as to accommodate
a wide range of cell sizes for incorporation into appropriately sized microwells. For example, optical
fibers having core diameters ranging from 1 .6 to 100 urn are commercially available from either
2 0 Galileo Electro-Optics Corp. (Sturbridge, MA) or Edmund Scientific (Barrington, NJ). In addition,

larger sizes are available by custom order. Thus, appropriately sized fibers can be utilized to study
such diverse cell sizes as E. coli, with a typical cell dimension of 0.7 to 1.5 urn, and mammalian
neurons, with a cell dimension of up to 150 urn.

In one embodiment, a fiber optic array, having a 1 mm outer diameter and 7 urn individual

2 5 fiber core diameters, available from Galileo Electro-Optics Corp. (Sturbridge, MA), was utilized. One

end of the fiber optic array was polished using a series of aluminum oxide lapping films 12, 9, 3, 1 , 0.3
urn available from Mark V Lab (East Granby, CT). The fibers were then sonicated for approximately 1
minute to remove any residue from the polishing procedure. The etching procedure was performed by
submerging the distal face of the fiber at a right angle in 700 uL of a buffered hydrofluoric acid

3 0 solution, comprising 100 uL hydrofluoric acid (50%), 0.2g ammonium fluoride, and 600 uL of

WO 99/45357 PCT/US99/04473

deionized water, for approximately 65 seconds. The fiber was then rinsed with deionized water and
sonicated for 1 minute to remove any salts that may have been formed during the etching procedure.
In this embodiment, the etch reaction time was tailored such that the well size was 7 microns in
diameter, 3 microns in depth, and approximately 90 fL in volume. Both scanning electron microscopy
5 (SEM) and atomic force microscopy (AFM) may be utilized to characterize etched microwells. In Fig.
2a, a typical microwell array formed by the etching procedure is shown in an AMF photomicrograph
(Digital Instruments Nanoscope Ilia, Santa Barbara, Ca). in Fig. 2, an oblique view of the microwell
array is provided. As shown in these figures, the microwells formed by this process are extremely
uniform due to the uniform characteristic structure of the fiber optic array.

10 B. Selection of Cell Types

Virtually any cell type and size can be accommodated in fabricating the sensor of the present
invention by matching the cell size to individual optical fiber optic core diameters. Virtually any
naturally occurring or genetically engineered (i.e. containing exogeneous nucleic acid) eukaryotic or
procaryotic cell type may be used, with plants, invertebrates, bacteria and mammalian cells, including,

15 but not limited to, green fluorescent protein mutants, primate, rodent and human cells and cell lines
being preferred, as well as mixtures of cell types.

In one embodiment, NIH 3T3 mouse fibroblast cells were employed. These cells are
typically 15-20 urn in size. Other cells types such as E. coli bacteria, 1 x 3 um, staphylococcus
bacteria, approximately 1 um, myoblast precursors to skeletal muscle cells, 15-20 um, neutrophil

2 0 white blood ceils, 10 um, lymphocyte white blood cells, 10 um, erythroblast red blood cells, 5 um,

osteoblast bone cells, 15-20 um, chondrocyte cartilage cells, 15-20 um, basophil white blood cells, 10
urn, eosinophil white blood cells, 10 um, adipocyte fat cells, 20 um, invertebrate neurons (Helix
aspera), 125 um, mammalian neurons, 4-140 um, or adrenomedullary cells, 13-16 um, melanocytes,
20 um, epithelial cells, 20 um, or endothelial cells , 15-20 um, may be utilized as well. Additional other

2 5 suitable cell types include, but are not limited to, tumor cells of all types (particularly melanoma,

myeloid leukemia, carcinomas of the lung, breast, ovaries, colon, kidney, prostate, pancreas and
testes), cardiomyocytes, endothelial cells, epithelial cells, lymphocytes (T-cell and B cell) , mast cells,
eosinophils, vascular intimal cells, hepatocytes, leukocytes including mononuclear leukocytes, stem
cells such as haemopoetic, neural, skin, lung, kidney, liver and myocyte stem cells, osteoclasts,

3 0 chondrocytes and other connective tissue cells, keratinocytes, melanocytes, liver cells, kidney cells,

WO 99/45357 PCT/US99/04473

and adipocytes. Suitable cells also include known research cells, including, but not limited to, Jurkat
T cells, NIH3T3 cells, CHO, COS, etc. A particularly useful source of cell lines may be found in ATCC
Cell Lines and Hybridomas (8 th ed., 1994), Bacteria and Bacteriophages (19 th ed., 1996), Yeast
(1995), Mycology and Botany (19 lh ed., 1996), and Protists: Algae and Protozoa (18 th ed., 1993),
available from American Type Culture Co. (Rockville. MD), ali of which are herein incorporated by
reference.

C. Dispersion of Cells in Microwells

Once the microweU size is tailored to accommodate specific cell sizes, the next step in
creating an array of cells is to randomly disperse cells into the microwells. Fig. 3 is a schematic
diagram showing the method for dispersing and depositing cells in the microwell array. Cell
populations are conventionally cultured with growth media which matches cell needs. Culture media
is formulated according to either recipes provided by cell line providers, journal articles or reference
texts. A particularly useful reference for media preparation is ATCC Quality Control Methods for Cell
Lines (2 nd ed.), American Type Culture Co. (Rockville, MD) which is herein incorporated by reference.
After culturing, cells are typically trypsinized using aseptic techniques to remove them from the cell
culture dish and suspend them in growth media.

In one embodiment, NIH 3T3 mouse fibroblast cells, available from American Type Culture
Collection (Rockville, MD) were utilized. The cells were randomly dispersed into the wells by removing
the adhered cells from the culture dish with a proteolytic enzyme, such as Trypsin-EDTA (0.05%
Trypsin, 0.53 mM EDTA.4Na), available from ; Gibco BRL (Grand Island, NY], using aseptic
techniques and placing the cells into a single-cell suspension in growth media, such as Dulbecco's
Modified Eagle Medium with 1% PenicillinStreptomycin, 1% L-Glutamine-200mM, and 10% fetal calf
serum, available from Gibco BRL.

To disperse cells into the wells, approximately 1 .5 ml of the cell suspension was concentrated
by centrifugation at 2000 RPM for 3.5 minutes. The supernatant is drawn off and the centrifuge tube
was tapped to resuspend the cells. The cell suspension was then added to a 1 .5 mm diameter
capillary tube. Prior to adding cells to the microwell array, the end of the fiber optic array which
contains the microwells was sonicated under vacuum in cell media for approximately 1 5 minutes to
flush and fill the microwells with media. The microwell end of the fiber was then inserted into the
capillary tube and secured in place with a thin strip of laboratory film. The capillary tube/fiber set-up

WO 99/45357 PCT/US99/04473

was then incubated in the vertical position for 1-2 hours allowing the suspended cells to settle into the
wells and adhere to the well bottom. The length of time required to fill the microwells is dependent
only on the amount of time required for the cells to adhere to the microwell bottom. Excess cells
which were not accommodated by a well were wiped away with a cotton swab wet with growth media.
5 Fig. 4 is an SEM photomicrograph of a single cell dispersed in an etched microwell.

Once they are positioned within the microwells, cells with typically attach to microwell
surfaces within 1-2 hours by protein contact. Cell culture media in the microwells may be periodically
replenished by exposing the distal surface of the fiber optic array to fresh media and allowing
nutrients to diffuse into the microwell cavities. Typically, cells will divide every twelve to fifteen hours.

1 0 While the size of the microwells tends to confine individual cells, the array will accommodate limited
cell splitting over time. Microwell volume will restrict cell splitting due to the well know cell
phenomenon of contact inhibition when cells are touching.
D. Establishing Cell Viability

In one embodiment, the cell viability in the wells was investigated using a pH indicator, 2'-7'-

1 5 bis-(2carboxyethyl)-5-(and-6-)-carboxyfluorescein (BCECF-AM) which has an excitation wavelength of
505 nm and an emission wavelength of 535 nm and is available from Molecular Probes (Eugene,
OR). The acetoxymethyl (AM) ester form of BCECF is non-fluorescent in solution. The BCECF-AM is
cell membrame permeant and passively enters the cell where, once inside the cell, the lipophilic
blocking groups are cleaved by non-specific esterases resulting in an increase in fluorescent intensity.

2 0 This increase in fluorescent intensity is indicative of the cell viability. Fig. 5 is a schematic

representation of one embodiment of a method used for establishing cell viability in a sensor array.

For viability tests, an array of encoded cells previously dispersed in microwells was immersed
in a 1 uM solution of BCECF-AM for 1 minute. The cell array was removed and rinsed thoroughly to
remove any residual dye. Fluorescent images of cell responses were acquired every 30 seconds

2 5 using a 1 .0 second acquisition time to monitor the increase in fluorescent intensity due to the pH of

healthy cells. Prior to viability measurements, a cell location template was generated through
excitation of the encoded cells, using the encoding method described herein, and the locations of
individual cells was superimposed on viability measurement images in order to identify the location of
healthy cells within the array. A fluorescence image of the encoded cells is shown in Fig. 6. In Fig. 7,

3 0 the fluorescent intensity response of the cells indicates a gradual increase as the lipophilic groups are

WO 99/45357 PCT/US99/04473

cleaved from the BCECF-AM dye within the cell. The fluorescent intensity response of the wells which
do not contain cells was negligible.

In a alternative embodiment, BCECF-Dextran, available from Molecular Probes, was used for
cell viability measurements. A 0.1 uM solution of the dye was added to cell media contained within
5 array microwells. BCECF requires excitation at two wavelengths, 490 nm and 440 nm, and the ratio
of the emitted light intensity at 530 nm for each wavelength is proportional to pH. This dye is
conjugated with a large Dextran group to prevent entry into the cell through the cell membrane. Thus,
BCECF-Dextran can monitor decreases in pH within the external cell environment due to cell
metabolism. Fig. 10 shows the average response of nine cells over time where the pH of the cell

1 0 environment gradually decreases due to cell metabolism.

In another embodiment a commercial cell viability assay, LIVE / DEAD® from Molecular
Probes (Eugene, OR), was employed. This assay provides a two-color fluorescence cell viability
assay based on intracellular esterase activity and plasma membrane integrity. Live cells are
distinguished by the enzymatic conversion of the cell-permeant non-fluorescent calcein AM to

1 5 fluorescent calcein, with an excitation wavelength at 495nm and an emission wavelength at 515nm.
Dead cells are distinguished by binding ethidium homodimer (EthD-1), with an excitation wavelength
at 495nm and an emission wavelength at 635nm, to nucleic acids which is accompanied by a 40-fold
increase in fluorescent intensity. EthD-1 is excluded by the intact plasma membranes of living cells.
Background fluorescence levels are inherently low with this assay technique because the dyes are

2 0 virtually non-fluorescent before interacting with cells.

In a typical procedure, a working solution of 2 uM calcein AM and 4uM EthD-1 was prepared
in serium-free medium. An array of N1H 3T3 mouse fibroblast cells was cultured at the distal face of a
microwell array. The proximal face of the imaging fiber was focused on the imaging system. The cell
array at the distal face of the imaging fiber was placed in serium-free medium without dye. The cell

2 5 array was excited at 495nm and emission values from a population of 25 cells were acquired for
300ms at 515nm (Live) and 635nm (Dead). These values serve as background reading for these
measurements. The cell array was then placed in the dye solution and the cell array was excited at
495nm and emission values from 25 cells were acquired for 300ms at 515nm (Live) and 635nm
(Dead) every minute for 30 minutes. Ail measurements were performed at room temperature.

WO 99/45357 PCT/US99/04473

Average fluorescent intensity for the live and dead cell wavelengths are plotted versus time after
subtracting the background fluorescence measurement at each emission wavelength.

This assay was used to evaluate pretreatment methods for filling the microwells of a
biosensor arrray with culture media prior to inserting cells. By comparing the viability of cells placed in
5 microwells after various treatments, it was determined that, in a preferred embodiment, sonicating the
mircrowell array under vacuum for 1 5 minutes prior to insertion of cells improves viability by ensuring
that the microwells are filled with culture media. In Fig. 11a, the average fluorescent intensity for dead
cells and live cells is plotted with time where no pretreatment was utilized. In Fig. 1 1 b, results are
plotted for a pretreated microwell array. Comparison of the two figures demonstrates the advantage

10 of prefilling the microwells with culture media prior to cell insertion. In this example, when the

microwell array was not sonicated prior to adding the cells the cells displayed immediate cell death.
In this example, when the microwells were sonicated under vacuum to fill the arrays with culture
media, the cells remained viable for approximately 20 minutes.
E. Encoding Cell Populations

15 A unique feature of the biosensor array of the present invention is that cells within each cell

population are individually encoded for maintaining cell identity within the array when randomly mixed
populations of cells are employed. Cells may be encoded with a single fluorophore or chromophore
dye or ratios of such dyes. Alternatively, cells may be encoded by either injecting a non-toxic
fluorescing compound into the cell cytoplasm or by employing natural or genetically-engineered cells

20 lines which exhibit chemiluminescence or bioluminescence , such as green fluorescent protein

mutants. Although a plurality of cell populations may be randomly mixed in the biosensor array, the
identity and location of each cell type is determined via a characteristic optical response signature
when the array is illuminated by excitation light energy. Cells may be encoded prior to disposing them
in the microwells or, alternatively, following placement in the microwells. . In one embodiment, a

2 5 single fluorophoric or chromophoric material or dye is used for encoding the cells. In an alternative

embodiment, two or more encoding materials or dyes may be used to encode cell populations and the
ratio of the optical response light intensity from each material or dye, produced by exposure to
excitation light energy, is used to identify the positions of individual cells within the cell population and
locate them in the array. In various alternative embodiments, cells may be encoded with dye

3 0 compounds which are attached to cells, taken up by cells or provided in the local cell environment.

WO 99/45357 PCT/US99/04473

A wide variety of fluorophores, chromophores, stains or a dye compounds may be used for
encoding cells. Encoding dyes may be permeant or impermeant to the cell membrane. Impermeant
dyes may be conjugated with acetoxymethyl ester to allow take up by cells. In one embodiment,
conventional conjugate or reactive cell membrane stains, cell tracers, or cell probes such as
5 fluoresceins, rhodamines, eosins naphthalimides, phycobiliproteins, nitrobenzoxadiazole may be
utilized. In other embodiments, cyanine dyes, such as SYTO® (Molecular Probes), amine-reactive
dyes, thiol-reactive dyes, lipopilic dyes, and DNA intercalators, such as acridine orange, may be
employed. In one embodiment, fluorogenic or chromogenic enzyme substrates may be taken up by
the cells, processesed by intracellular enzymes, such as glycosidases, phosphatases, luciferase, or

10 chloramphenicol acetyltransferase, and provide encoding for cell populations. In an alternative
embodiment, cell organelle dye probes may be employed for encoding. In one embodiment, cell
membrane probes such as carbocyanines and lipophilicaminostyrls may be utilized for encoding.

By way of example, Tables 1 and 2 provide a partial listing of a various types of dyes and
their corresponding excitation and emission wavelengths which have utility for encoding cell

15 populations in sensor arrays of the present invention. In addition, a particularly useful reference for
selecting other types of encoding dyes is R.P. Haugland, Handbook of Fluorescent Probes and
Research Chemicals (6 th ed.). Molecular Probes lnc.(Eugene, OR, 1996).

Cell encoding eliminates the need for mechanically scanning the array, mechanically indexing
the location of cells, and mechanically positioning the array for measurements of individual cells. In

2 0 addition, by encoding all cells within the array, simultaneous measurements of the entire array are
possible since the responses of individual cells are readily associated with a specific cell population
type because each cell is encoded. Encoding of cells thus provides for rapid, simultaneous
measurements of all cells and cell populations within the array without the need to mechanically scan
the array to acquire a series of sequential measurements for each cell. Since monitoring and

2 5 measuring the responses of all cells in the array occurs simultaneously, without a prolonged delay
between the first cell measurement and last cell measurement, the biosensor array of the present
invention provides the capability to monitor both short term and long term cell responses to biological
stimuli. The biosensor array of the present invention thus has the unique ability for measuring both
cell responses and response rates to analytes. This feature provides

WO 99/45357

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26
TABLE 1

1 . Plasma Membrane Stains

5 PKH22 green fluorochrome 551ex/567em
PHK67 green fluorochrome 490ex/502em
PKH26 red fluorochrome 551ex/567em

lipophilic carbocyanines

Dil - orange fluorescence
DiO - green fluorescence
DiD - red flurescence

FM 1-43 - green fluorescence 510ex/626em
15 FM 4-64 - red fluorescence 51 5ex/640em

RH 414 - orange fluorescence 532 ex/71 6em

lipophilic dextrans

tetramethylrhodamine dextrans (10,000 MW) 470ex/500em

2 0 fluorescein dextran (10,000 MW) 495ex/530em

2. Tracker Probes

Blue 7-amino-4-chloromethyl coumarin 354ex/466em
25 4-chloromethyl-6 t 8-difluoro-7-hydroxy coumarin 371ex/464em

4- chloromethyl-7-hydroxy coumarin 372ex/470em
Green 5-ch I oromethyl fluorescein diacetate 492ex/516em
8-chloromethyl BODIPY 522ex/528em

3 0 Yellow-Green

5- chloromethyleosin diacetate 524ex/544em

Orange

5-(and 6H((4-chloromethyl)benzoyl)amino) tetramethylrhodamine 540ex/566em

3. Amine-Reactive Probes

fluorescein isothiocyanate 494ex/519em
carboxyfluorescein succinimidyl esters 504ex/529em

4 0 carboxyeosin succinimidyl esters 492ex/517em

WO 99/45357 PCT/US99/04473

additional discriminating response information which is useful for detecting biological or chemical
analytes of interest.

While a number of encoding dyes and methods are available, in one embodiment, external
fluorescent cell membrane labels , PKH67 with an excitation wavelength of 490 nm and an emission
5 wavelength of 502 nm, and PKH26 with an excitation wavelength of 551 nm and an emission

wavelength of 567 nm, were utilized. PKH26 and PKH67 are part of a family of dyes manufactured by
Zynaxis Cell Science (Malvern, PA), sold under the trademark Zyn-Linker® (Phanos Technologies
Inc.), produced by the method of U.S. Patent 5,665,328 to Hogan, et a!., and available from Sigma
(St.Louis, Mo). In this embodiment, both dyes were applied to suspended fibroblast cells to encode

10 the cells prior to placement in the sensor array. Encoded cells were placed in microwells filled with
growth media. The encoded cells were then illuminated with excitation light transmitted through the
fiber optic array and the resulting fluorescent emission response of cells was collected through the
same fiber and passed to the detection system. Fig. 6 shows a typical fluorescence optical image of a
PKH26 encoded cell population in a biosensor array of the present invention. Fig. 8 shows a typical

15 fluorescence image of a PKH67 encoded cell population in a biosensor array. Note that only those
microwells containing encoded cells yields a fluorescent signal upon excitation at the encoding dye
wavelength.

In a typical encoding procedure, external or internal encoding labels are applied to suspended
cell populations prior to placement in the sensor array. In one embodiment, an external label is
2 0 applied to the cell by first washing the suspended cells with serum-free media and then centrifuging
the cells into a loose pellet at 2000 RPM for 5 minutes. The supernatant is drawn off and the
centrifuge tube is tapped to resuspend the cells. Approximately 1 mL of Diluent C, available as dye
kit from Sigma, was added to the cells and the tube was inverted to mix. Immediately prior to
encoding, a solution of 4 x 10" 6 M Diluent C dye was prepared. The suspended cells are added to the

2 5 dye solution, mixed by pipetting the cell/dye solution, and incubated for 5 minutes. To stop the

encoding reaction, 2 mL of fetal calf serum was added to the cells, incubated for 1 minute, and then
diluted by adding 4 mL growth media. The cells are then centrifuged at 2000 RPM for 10 minutes to
remove the cells from the staining solution. The supernatant was drawn off and the cells are
transferred to a new tube. Finally, the cells are subjected to a minimum of three washings by adding

3 0 10 mL growth media, centrifuging, and resuspending the cells.

WO 99/45357

PCTYUS99/04473

Chapter 1 2 — Section 12.2 Celt-PermeMnt Pmfw»« wwiw,^.

WO 99/45357 PCT/US99/04473

To demonstrate the encoding method with PKH26 and PKH67 dyes, encoded NIH 3T3
mouse fibroblast cells were dispersed in microwells in sensor array and the location of encoded cells
within the array was determined by excitation of the cells at a wavelength of 551 nm. Upon excitation,
the encoded cells form a characteristic, detectable fluorescence pattern within the sensor array where
5 the pattern provides a locational template for cell viability measurements using BCECF and an

excitation wavelength of 505 nm. The excitation and emission wavelengths of the PKH26 encoding
label and the BCECF-AM dye are sufficiently separated so that there is no interference between the
two dyes. Fig. 6 is a characteristic fluorescence image pattern identifying the location of PKH26
encoded cells in the biosensor array. A similar procedure was followed for encoding the mouse
1 0 fibroblast cells with PKH67 where a corresponding characteristic fluorescence image pattern is shown
in Fig. 8.

In addition to encoding individual cell populations with a single unique dye, each cell
population within the array may be encoded with unique dye ratios which yield a characteristic
fluorescent intensity ratio in a distinct wavelength range. A range of dye ratios may be employed with

15 two or more dye combination for producing a series of encoding ratios useful for a identifying a large
number of cell populations. The fluorescent intensity ratio, produced by a specific dye ratio which is
used for encoding a cell population, can be distinguished by taking the mean intensity minus the
background intensity at each emission wavelength and then dividing the two values to obtain the
range for that particular ratio.

2 0 In one embodiment, two separate populations of NIH 3T3 mouse fibroblast cells were

encoded by labeling cell membranes with either a 1:5 ora 5:1 ratio of PHK67 and PHK26 dyes.
Each cell population comprised approximately 125 encoded cells. The cell populations were
illuminated at 490 nm and 551 nm excitation wavelengths and emitted fluorescent intensity ratios
were measured at 502 nm and 567 nm. Measurements of the average intensity ratio for each dye

2 5 ratio were made over a two day period. The initial average intensity ratio for the 1 :5 PKH67/PKH26
ratio was 0.0863 and the ratio for the 5:1 dye ratio was 0.7014. The final average intensity ratio for
the 1 :5 ratio was 0.2655 and for the 5:1 ratio it was 0.9090 indicating that the ratios are reasonably
stable, even where cell splitting has occurred, and that dye ratio encoded cell populations remain
distinguishable with time. Thus, dye ratios can provide a useful alternative encoding mechanism for

WO 99/45357 PCT/US99/04473

identifying and locating cell populations which are randomly dispersed on a biosensor array of the
present invention.

F. Indicator Dyes

The optical responses of individual cells and cell populations to chemical or biological stimuli
5 are typically interrogated and detected by coupling individual cells with appropriate indicators which
may be either fluorophores, chromophores, stains or a dye compounds. Any suitable indicator or
combinations of indicators may be utilized provided the indicator does not compromise cell response.

For example, conventional cell fluorophore probes such as fluoresceins, rhodamines,
naphthalimides, phycobiliproteins, nttrobenzoxadiazole may be utilized. Alternatively, permeant or
10 impermeant cell membrane potential indicators, ion indicators, reactive oxygen indicators and pH

indicators may be employed. By way of example a number of indicators which would have particular
utility for the biosensor array of the present invention are listed in Tables 3 through 8 together with
their characteristic excitation and emission wavelengths. A particularly useful reference for selecting
appropriate indicators is R.P. Haugland, Handbook of Fluorescent Probes and Research Chemicals
15 (6 th ed.), Molecular Probes lnc.(Eugene, OR, 1996).

In one embodiment, indicators may be incorporated directly into the cell by attachment to the
cell membrane, by absorption into the cell cytoplasm by membrane permeant indicators, or by
microinjection into the cell cytoplasm. In an alternative embodiment, ultrafine fluorescing
microspheres, such as Molecular Probes FluoSpheres™ , are ingested by the cells and are employed
2 0 as indicators. In another environment, indicators are added to the culture media fluid contained within
the microwells. In an alternative embodiment, indicators may be attached to the surface of the
microwells by a conventional

WO 99/45357 PCT/US99/04473

PAGE INTENTIONALLY LEFT BLANK

WO 99/45357

PCT/US99/04473

pH Range

Typical Muiurmtii ]

SNAFLtadkatora

7.2~«.2

Excitation ratio 4907540 nm *♦ ]
emission ratio 540/630 nm |

SNARF indicators

7.0-8.0

Emission ratio 580/640 mi.

HPTS <pyreninc)

7.0-8.0

Excitation ratio 430/405 im.

BCECF

6.5-7 5

ExciUiiion ratio 490/44O m.

Fluoresceins and
carboxyfluoresceuu

6.0-7.2

Excitation ratio 490/45n n»

Oregon Green dyes

4.2-5.7

Excitation ratio 5 1 0/4 SO im •
excitation runo 490/440 mi.

RhodoU (including
NERF dyes)

4.0-6.0

Ear iuiion ratio 5 14/48* nm . •
excitation ratio 500/450 nm

LysoSetuor probei

3.3-6.0 •

Excitation ratio 340/380 nm •

• Depends on pK. oT Mtacted prate: bm Table
probe, f Applies to L-7S43 only. Oihrr Ly*o3

23 .2 tor pK, of «»ch Ly*oS*n*«
*mor probes allow tmgk iuhm>.
2 lot wsvetcofthi.

Tmhtt 23 J Summary of the pH response of our LytoStnsor ptnhes.

Cat*

Ptobe

Abakan*

Useful pH
Range t

L-7532

LyaoSanaor
BhwDKlVt92

374M34

65-6.0

U73J3

Bum»NI>167

373*23

4.5-6.0

U7534

LyaoSanaor Omen
DNO-153

442/505

63-8.0

L-7535

LyaoSanaor Oraen
DND-1B9

443/505

5.2

4 5-60

U7545

LyaoSanaor
Yellow/Blue
DND-160

3*4/540*
3297440 1

4.2

3.0-5.0

'Anwrfaioa CAM) aniftiinwi rear ■ eanuiuii (Em)iaaiima «»*t3. value* may vary
■a— east im snUalsi eaeiiBsaeaaii t AM pK, whan iw a aaanene d w ww. yajaat
- • ^**~*mealK iets40;|atpH7

-e 3

ToWe 2J.4 Re derive pH inu.catQf<iyes.

} pH Indicator

Prefetrad lUactirc Form

BCECF

BCECF (B- 1 1 5 1 , see Section 23 J) *

1 CiirhnkynuorvRCcin

5-(and-6)-cartx»xyfUwreacein,
noccinimidy 1 asiar (C- J 3 1 1 , aee Section 1 .3)

CI-NbRF

Cl-NERF (C-6831. tee Section 23.3)*

DichHiroOuorcvcetn

2',7'-dichloronuore»cein-3-l»othiocyanal«
(D-6078. tee Section 1.3)

JJ Dimethylfluorescein

>-t ano -o ) -caroo* y-* .3 -ojincmyuioorcnoBin
<C-366.see Section 23.2)*

It r\KA WPDF

|i UM'Nr.Kr

DM.NRRF /TVABV). tee Section Tk3\ *

II N;iphih«inuorescein

3 •( end -6 >-carbo*yn tphthofluoreacsin,
luccinimidyl atter (C-653)

| < hrjinn < ireen 4KH

Ore aim Green 4M carboxylic acid,
Micclnimidyl enter (0-6I47, 0-6149)

Orccon Green 300

Oregon Green 500 carboxylic acid,
succinimidyl «*» <0-6136)

Oregon Green 514

Oregon Green 514 carboxylic acid,
succ.mmidyl cater (0-6I39)

RhiMktl Green

RhoUol Orean carboxylic acid*

succinimidyl ceaer (R.6I06) 1

I SNAH..I

5~Und^)<afboay SNAFU,
mice inimidyl eater <C*306I )

SNAIL- 2

V(»nd-6>-earboiy SNAFU-2,
auccinimidyl eater (C-3062)

SNARF- 1

5-(und-6>-carboxy SNARF- 1
(C- 1270. tee Section 23.2) •

(>c»- Scvi ion l.t t.

WO 99/45357

PCT/US99/04473

WO 99/45357 PCT7US99/04473

— I

fcMfr tt.Y Summary of fluorescent CV ' muu iifori umtlante from Molecular Probes

J Ca* Indicator

Salt * ! AM Eittr ♦

Deatran J

Model

K.(nM)t

Now*

1 BU-TUTl

B-6810

Ex 34(1/3 HO

370

)

B-6790 | B-6791

Ex 400/4BO

7000

1 Calcium Green* 1

O30I0 : C.Ulll.C-3012

C-6765. C 37I3.
C.37-.4.C-b766

Em 530

190

1 Calcium Oraen-2

C-3730 | IVI7U

Em 533

550

3.1

1 Calcium Green -3 N

C-3737 j CW7J9

Em 530

14.000

| Calcium Orange

C-3013 CYMI3

Em 575

185

| Calcium Orunge-SN

C-6770 : C-A77I

Em 580

20.000

1 Calcium Crimaon

C-3016 ' C-3UIH

C-6824. C-6825

185

| Ruo-3

F-I24H.F 3715

I--I24I. F- 1 242

Em 525

390

3.4

1 Fuiu2

F-I2W1. F-6799

I- I2U1. F-I221.F-1223

F-6764. F-3029.
F-3030

E« 340/380

145

1 Fun Red

F-3019

I-.1020.F- 3021

E% 420/4H0

140

2.6.7

1 lndo-1

I- 1202

U203.M223.M226

1-3032. 1-3033

Em 405/483

230

1 a4eg-fufe-2

M-1290

M-I291.M-1292

E* 340/380

25J000

1 Mag-fura-5

M-3103

M-3105

E» 340/380

28.000

1 Magmdo-l

M-1293

M-6907, M-690S

Em 405/485

35.000

2.8

| ftiejnseium Green

M-3733

M-3735

Em 530

6000

1 Onion Green 488 BAPTA- 1

O-6H0fi

O-AK07

0-6798. 0-6797

tm520

170

)

[oregon Green 488 BAPTA 2

OftMUX

OfiKlN

Im520

580

3.V

■ Cetgon Green 488 BAFTA-5N

0-6K12

Ij«520

20.000

0-I2H7

O-12K*.0-i289

\ jm 495

2.10

ltfcod-2

R 1243

R-J 244. R.I 243

lim 570

570

C-6800

lim 535/615

370

i.C«^«*«*»«l«^«n«.-h. t CM*-* AM— •■ , Cuw «*» 7^3^, J„^^^^^ > ^ , •^

I «al HaWOW, 2. AM ewer loan .» nuoretcou (. injur putemi.1 Murct of em« .nC.'- mewiMtmtn.'
1 Lla^fewCa^-dBpmdMU AuorciceiKe fOkponsc to luri, » hvi * «"-* cr ^""7"'"': - _ lfWt r.:-.!,-. IO rrm fcutmtuOe of C* 1 ' -dependent ihiwaaccsK* inert**
1 7 --- *««I^Md*MM. ac .ni- a Calcium Graci^ 1 ♦» i«o« rtuureuem U«Jn rtue-3 m buih C« -hound and t ■ •me lonm. «JT.i-»^h nun MttumN.
I kAM our forni » nonnuomecni. *■ w« m ' J "* r r ..,,,„_ <;mgn , . ^ L ari te UBe4 j 1R combination with fluw-3 for 6uai-w»**ionfUi ratio nowimwu,

Chapter 22

Section 22.1 Introduction to Ca 2 + Measurements with Fluorescent Indicators

WO 99/45357 _

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PCT/US99/04473

#h - ?W fir* ~ J/7

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itAA^^c^i Ah ■ WS" - V//

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5*0/3*0

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■70 /

WO 99/45357 PCT/US99/04473

J - ft**: tf' 3H6/9SO /

7«y - Undc • / *o r/yfcr /

56F0 35^1 61tT

SkIuxvo (jAJJUf^ 3)6/ 'S33" ~ VLUUi 2i )iJ &t . - e*mJC&.<-l f Oti>H/y? edicts.

1~ cc*h> s~S

WO 99/45357

PCT/US99/04473

dtktis

F( di/Sufsi- 6reen \ aiMiu tiursf ^v*/^
Oi Chiorod> hd rc ^luoresctt /) DiazfUk - /ncm&rs OX/M/W acdi/,'//

SDW / SZ«? O.X III

Di hydro rhodamirw 12.^ - i/)Mskfiks nackvl exyyn /flttrYVvrf/<tfcs
e^5D7/529«K prodvx.(xcl l<x| "pho^oc^-lts . — ^J.

Rmtx. Rad ™ - ca\cuxw\ irtfltcocW" - mmfimeJ ^WaU^Art — borate
e** s/s/ 60S*

WO 99/45357 PCT/US99/04473

silanization treatment for bonding the indicator to the glass surface. In one embodiment,
natural or genetically engineered ceil lines which exhibit chemiluminescence, bioluminesence, or color
changes when stimulated are used alone without need for a separate indicator since they produce
intrinsic optical responses. Examples of such cells include green fluorescent protein mutants. In
5 other embodiments, cells which express enzymes are employed with a reagent which enables an
optical response, such as the generation of a fluorescent product. For example when luciferase is
expressed by a cell in the presence of luciferen, the cell produces a fluorescence response when
biologically stimulated.

In alternative embodiments, multiple indicators may be employed within a cell array, within a
10 cell population, or within individual cells so as to provide for simultaneous monitoring of cell responses
to a variety of different chemical or biological materials. Where two or more indicators are utilized,
dyes with spectral separation of narrow spectral bandwidths are particularly useful, for example
Molecular Probes BODIPY™ dyes.
G. Optical Measurements

15 Individual cells and populations of cells within the biosensor array of the present invention

may be optically interrogated and ceil responses may be monitored by employing conventional optical
components known to one of ordinary skill in the art. Where external optical stimulation of cells is
required to elicit an optical response, conventional light sources such as arc lamps, photodiodes, or
lasers may be employed for excitation light energy. Cell responses may be monitored by

2 0 conventional detectors such as photomultiplier tubes, photodiodes, photoresistors or charge coupled
device (CCD) cameras. Conventional optical train components, such as lenses, filters, beam splitters,
dichroics, prisms and mirrors may be employed to convey light to an from such light sources and
detectors either to discrete substrate sites or through optical fiber strands to microwells that contain
individual cells. The principal requirement for any particular optical apparatus configuration that is

2 5 employed in optical measurements is that the combination of optical components provide for optically
coupling the cells in the array to detectors and light sources. While a particular apparatus
configuration that was employed in experimental optical measurements is described below, other
configurations may also be employed which are functionally equivalent and appropriate suited for a
particular measurement requirement.

WO 99/45357 PCT/US99/04473

The instrumentation used for fluorescence measurements is a modified Olympus (Lake
Success, NY) epifluorescence microscope which was converted from a vertical to a horizontal
configuration. A schematic diagram of the measurement system 100 is shown in Fig. 9. White light
from a 75W xenon lamp 1 10 is collimated by a condensing lens 1 12, passed through an excitation
5 filter 120, reflected by a dichroic mirror 130, and focused onto the proximal end 210 of an imaging
fiber 200 with a microscope objective 140. A neutral density filter 122 may be employed for
adjustment of excitation light intensity. The imaging fiber 200 is precisely positioned by an x-y
micropositioner 150, available from Spindler and Hoyer (Milford, MA), and a microscope z-translation
stage 1 52. Excitation light is transmitted through the fiber 200 to the biosensor array 300 at the distal

10 fiber end 212. The emitted fluorescence light from the biosensor array is returned through the fiber
200, through the dichroic mirror 130, filtered through an emission filter 160, and detected by a PXL™
Photometries (Tuscon, AZ) charge coupled device (CCD) camera 170. A magnification lens 165 may
be employed if necessary. Data is processed on an Apple Power Macintosh 440 (Sunnyvale, CA)
desktop computer 180 using IPLab 3.0 image processing software, commercially available from

15 Signal Analytics (Vienna, VA).

While a bench-top measurement system was utilized for these measurements, in one embodiment, a
compact portable measurement system may be assembled from conventional optical and electronic
components.

H. Biosensor Array Applications

2 0 The biosensor, biosensor array, sensing apparatus and sensing method of the present

invention can be applied to a large variety of conventional assays for screening and detection
purposes. The biosensor may be configured for virtually any assay and offers a distinct advantage for
high throughput screening where a plurality of encoded cell populations, which have utility in particular
assays or are genetically engineered cell to provide unique responses to analytes, may be employed
25 in a single sensor array for conducting a large number of assays simultaneously on a small sample.
The biosensor array thus provides both for tremendous efficiencies in screening large combinatorial
libraries and allows conduction of a large number of assays on extremely small sample volumes, such
as biologically important molecules synthesized on micron sized beads. The biosensor of the present
invention can be applied to virtually any analyte measurements where there is a detectable cell

3 0 response to the analyte due to biological stimulation.

WO 99/45357 PCT/US99/04473

The biosensor array and method of the present invention utilizes the unique ability of living
cell populations to respond to biologically significant compounds in a characteristic and detectable
manner. Since the selectivity of living cells for such compounds has considerable value and utility in
drug screening and analysis of complex biological fluids, a biosensor which makes use of the unique
5 characteristics of living cell populations offers distinct advantages in high throughput screening of
combinatorial libraries where hundreds of thousands of candidate pharmaceutical compounds must
be evaluated. In addition, such a biosensor and sensing method can be utilized for either off-line
monitoring of bioprocesses or in situ monitoring of environmental pollutants where the enhanced
sensitivity of living cells to their local environment can be exploited.

10 Thus, the present invention provides methods for detecting the responses of individual cells to

analytes of interest. By "analyte of interest" or "target analyte" or "candidate bioactive agent" or
"candidate drug" or grammatical equivalents herein is meant any molecule, e.g., protein, oligopeptide,
small organic molecule, polysaccharide, polynucleotide, etc., to be tested for the ability to directly or
indirectly altering a cellular phenotype, including its optical properties. Generally a plurality of assay

15 mixtures are run in parallel with different agent concentrations to obtain a differential response to the
various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at
zero concentration or below the level of detection.

Analytes encompass numerous chemical classes, though typically they are organic
molecules, preferably small organic compounds having a molecular weight of more than 100 and less

2 0 than about 2,500 daltons. Analytes comprise functional groups necessary for structural interaction
with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl,
hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate
agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic
structures substituted with one or more of the above functional groups. Candidate agents are also

2 5 found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines,

derivatives, structural analogs or combinations thereof. Particularly preferred are peptides.

Candidate agents are obtained from a wide variety of sources including libraries of synthetic
or natural compounds. For example, numerous means are available for random and directed
synthesis of a wide variety of organic compounds and biomolecules, including expression of

3 0 randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial,

WO 99/45357 PCTAJS99/04473

fungal, plant and animal extracts are available or readily produced. Additionally, natural or
synthetically produced libraries and compounds are readily modified through conventional chemical,
physical and biochemical means. Known pharmacological agents may be subjected to directed or
random chemical modifications, such as acylation, alkylation, esterification, amidification to produce
5 structural analogs.

In a preferred embodiment, the candidate bioactive agents are naturally occuring proteins or
fragments of naturally occuring proteins. Thus, for example, cellular extracts containing proteins, or
random or directed digests of proteinaceous cellular extracts, may be used. In this way libraries of
procaryotic and eucaryotic proteins may be made for screening in the methods of the invention.

10 Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian
proteins, with the latter being preferred, and human proteins being especially preferred.

The peptides may be digests of naturally occuring proteins as is outlined above, random
peptides, or "biased" random peptides. By "randomized" or grammatical equivalents herein is meant
that each nucleic acid and peptide consists of essentially random nucleotides and amino acids,

15 respectively. Since generally these random peptides (or nucleic acids, discussed below) are

chemically synthesized, they may incorporate any nucleotide or amino acid at any position. The
synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the
formation of all or most of the possible combinations over the length of the sequence, thus forming a
library of randomized candidate bioactive proteinaceous agents.

20 In one embodiment, the library is fully randomized, with no sequence preferences or

constants at any position. In a preferred embodiment, the library is biased. That is, some positions
within the sequence are either held constant, or are selected from a limited number of possibilities.
For example, in a preferred embodiment, the nucleotides or amino acid residues are randomized
within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased

2 5 (either small or large) residues, towards the creation of nucleic acid binding domains, the creation of

cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for
phosphorylation sites, etc., or to purines, etc.

As described above generally for proteins, nucleic acid candidate bioactive agents may be
naturally occuring nucleic acids, random nucleic acids, or "biased" random nucleic acids. For

3 0 example, digests of procaryotic or eucaryotic genomes may be used as is outlined above for proteins.

WO 99/45357 PCT/US99/04473

"Nucleic acids" in this context includes both DNA and RNA, and nucleic acid analogs including PNA.In
a preferred embodiment, the candidate bioactive agents are organic chemical moieties, a wide variety
of which are available in the literature.

While the examples below provide a variety of specific assays which may be useful in
5 configuring and employing the biosensor array and method of the present invention, they are not
intended to limit either the scope of applications envisioned or the broad range of sensing methods
which can be employed with a plurality of cell populations with the biosensor of the present invention.

In one embodiment, the biosensor array can be employed for remotely monitoring redox
states of individual cells or cell populations in bioprocesses. For example, NADH dependent
1 0 fluorescence can be measured in bacteria, fungi, plant or animal cells. NAD(P) /NAD(P)H can be

measured to monitor changes from aerobic to anaerobic metabolism in fermentation processes using
the method disclosed by Luong, et al., in Practical Fluorescence . G. Guilbault ed., Marcel Dekker
(New York, 1990).

Alternatively, the biosensor array may be employed for in situ monitoring of cellular
15 processes in response to environmental contaminants by incorporating the method disclosed by

Hughes, et al. a Analytics Chimica Acta 307:393(1995) to provide for distinguishable cell population
responses within an array. In this method, micron-sized spheres, impregnated with a fluorophore and
modified on the surface with a fluorogenic enzyme probe, are ingested by cells and enzymatic activity
occurs at the sphere surface, producing a detectable fluorescent signal.
20 In yet another embodiment, the biosensor array can be employed with genetically engineered

bioluminescent bacteria for in situ monitoring and optical sensing of metallic compounds. For
example, cell population responses to antimonite and arsenite may be utilized by incorporating the
method disclosed in Ramanathan, et al.. Anal Chem. 69:3380(1997) into cell populations within the
biosensor array. With this method, cell plasmid regulates the expression of bacterial luciferase

2 5 depending on the metal concentration.

In another embodiment, the cell populations within the biosensor array can be encoded with
ATP dependent luminescent proteins, for example firefly luciferase, which are injected into rat
hepatocytes for pathological studies according to the method disclosed by Koop, et a!., Biochem. J.
295:165(1993). These cells exhibit a decrease in cytoplasmic ATP when exposed to pathological

3 0 insults and changes in fluorescence directly relate to the extent of metabolic poisoning in the cell.

WO 99/45357 PCTAJS99/04473

In one embodiment, the cell populations within the biosensor array can be encoded with
green fluorescent protein [see T. Gura, Science 276:1989(1997); Niswender, et al. f J. Microscopy
180(2):1 09(1 995); Cubitt, et al. f TIBS 20:448(1995); Miyawaki, et al., Nature 388:882(1997)]. Several
genetically-engineered mutants of GFP are available which have distinguishable fluorescence
5 emission wavelengths. These proteins have additional utility as fluorescing indicators of gene
expression and Ca* levels within cells.

In an additional embodiment, the biosensor array can be used in measurements of cell
proliferation by in situ monitoring of calcium levels and calcium oscillations in single cells using
fluorescent markers, such as aequorin orfura-2, according to the method disclosed by Cobbold, et al.,

1 0 Cell Biology 1:311 (1 990).

As will be appreciated by those in the art, the assays of the invention may be run in a wide
variety of ways and for a wide variety of purposes. For example, the cells may be used as a detection
system for a particular analyte; the cells undergo a characteristic optically detectable change in the
presence of a particular analyte. Alternatively, the cells may be used to screen drug candidate

15 libraries for the ability to alter a cellular phenotype that is optically detectable. For example, the

expression of a therapeutically relevant cell surface receptor may be increased such that the receptor
can now bind a fluorescent ligand; similarly a therapeutically relevant enzyme may now be activated
such that a fluorescent reaction product is generated. In this way any modulation, induing both
increases and descreases, may be monitored. Similarly, the use of reporter genes such as green

2 0 fluorescent proteins and derivatives thereof facilitates high throughput screening for relevant analyte
interactions, through the use of inducible promoters, for example.

Generally, in a preferred embodiment, a candidate bioactive agent is added to the cells prior
to analysis, and the cells allowed to incubate for some period of time. By "administration" or
"contacting" herein is meant that the candidate agent is added to the cells in such a manner as to

2 5 allow the agent to act upon the cell, whether by uptake and intracellular action, or by action at the cell
surface. In some embodiments, nucleic acid encoding a proteinaceous candidate agent (i.e. a
peptide) may be put into a viral construct such as a retroviral construct and added to the cell, such
that expression of the peptide agent is accomplished; see PCT US97/01019, hereby expressly
incorporated by reference.

WO 99/45357 PCT/US99/04473

Once the candidate agent has been administered to the cells, the cells can be washed if
desired and are allowed to incubate under preferably physiological conditions for some period of time.

The reactions outlined herein may be accomplished in a variety of ways, as will be
appreciated by those in the art. Components of the reaction may be added simultaneously, or
5 sequentially, in any order. In addition, the reaction may include a variety of other reagents may be
included in the assays. These include reagents like salts, buffers, neutral proteins, e.g. albumin,
detergents, etc which may be used to facilitate optimal detection, and/or reduce non-specific or
background interactions. Also reagents that otherwise improve the efficiency of the assay, such as
protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used.

10 In general, at least one component of the assay is labeled. By "labeled" herein is meant that

the compound is either directly or indirectly labeled with a label which provides a detectable signal,
e.g. radioisotope, fluorescers, enzyme, antibodies, particles such as magnetic particles,
chemiluminescers, or specific binding molecules, etc. Specific binding molecules include pairs, such
as biotin and streptavidin, digoxin and antidigoxin etc. For the specific binding members, the

15 complementary member would normally be labeled with a molecule which provides for detection, in
accordance with known procedures, as outlined above. The label can directly or indirectly provide a
detectable signal.

Once the assay is run, the data is analyzed to determine the experimental outcome, i.e. either
the presence or absence of a target analyte, the effect of a candidate agent on a cellular phenotype,
2 0 etc. This is generally done using a computer.

In this way, bioactive agents are identified. Compounds with pharmacological activity are
able to alter a cellular phenotype. The compounds having the desired pharmacological activity may
be administered in a physiologically acceptable carrier to a host, as previously described. The agents
may be administered in a variety of ways, orally, parenterally e.g., subcutaneously, intraperitoneally,

2 5 intravascular^, etc. Depending upon the manner of introduction, the compounds may be formulated

in a variety of ways. The concentration of therapeutically active compound in the formulation may
vary from about 0.1-100 wt.%.

The pharmaceutical compositions can be prepared in various forms, such as granules,
tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like. Pharmaceutical grade

3 0 organic or inorganic carriers and/or diluents suitable for oral and topical use can be used to make up

WO 99/45357 PCT/US99/04473

compositions containing the therapeutical ly-active compounds. Diluents known to the art include
aqueous media, vegetable and animal oils and fats. Stabilizing agents, wetting and emulsifying
agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin
penetration enhancers can be used as auxiliary agents.

While this invention has been particularly shown and described with references to preferred
embodiments thereof, it will be understood by those skilled in the art that various changes in form and
details may be made therein without departing from the spirit and scope of the invention as defined by
the appended claims.

WO 99/45357

PCT/US99/04473

CLAIMS

1. A biosensor for detecting the response of individual cells to at least one analyte of interest
comprising:

5 a) a substrate comprising a plurality of discrete sites; and

b) a plurality of cells each dispersed at one of said discrete sites, wherein each cell is
encoded with at least one optically interrogatable material.

2. The biosensor of claim 1 wherein said substrate comprises a fiber optic array comprising a

10 plurality of fibers, each fiber having a distal end and a proximal end, and said discrete sites comprise
a plurality of microwells each formed on an end surface ofsaid distal end, and each one of said
plurality of cells is dispersed in one of said microwells, wherein each of said cells is optically coupled
and in optical

communication with a distal end of at least one of said fibers in said array.

3. The biosensor of claims 1 or 2 wherein said sites have an internal diameter ranging from about 1
urn to about 200 urn, a depth ranging from about 0.25 urn to about 200 urn, and a volume ranging
from about 1 fL to about 5 nanoliters.

2 0 4. The biosensor of claims 2, or 3 wherein said fibers further comprise a fiber core having a diameter

ranging from about 1 urn to about 200 urn.

5. The biosensor of claims 1, 2, 3 or 4 wherein a surface of said sites are coated with a biologically
compatible material.

6. The biosensor of claims 1,2,3, 4, or 5 wherein an indicator compound is attached or inserted into
at least one of said cells.

7. The biosensor of claims 1 . 2, 3, 4, 5, or 6 wherein said plurality of cells comprises at least two cell

3 0 populations.

WO 99/45357

PCT/US99/04473

8. The biosensor of claim 7 wherein each cell population is encoded withat least one optically
interrogatable materials selected from the groupconsisting of a dye, a fiuorophore, a chromophore, a
chemiluminescent compound and a bioluminescent compound.

9. An apparatus for detecting the response of individual cells to at least
one analyte of interest comprising:

a) a biosensor array comprising:

i) a substrate comprising a plurality of discrete sites; and
10 ii) a plurality of cells each dispersed at one of said discrete sites, wherein each cell is

encoded with at least one optically interrogatable material.

b) a detector means optically coupled to and in optical communication with said discrete sites
on said substrate, said detector means capable of detecting an optical response of said cells

dispersed in said discrete sites to an analyte.

10. The apparatus of claim 9 wherein

said substrate comprises a fiber optic array comprising a plurality of fibers, each fiber having
a distal end and a proximal end;

said discrete sites comprise a plurality of microwells each formed on an end surface ofsaid
2 0 distal end of said fibers;

each one of said plurality of cells is dispersed in one of said microwells; and
said detector means is optically coupled to and in optical communication with a proximal end
of said fibers for detecting an optical response of said cells at said distal fiber ends.

2 5 1 1 . An apparatus according to claims 9 or 10, further comprising an excitation light energy means
coupled to an end of said fibers.

12. An apparatus according to claims 9, 10 or 1 1 further comprising an image capturing means for
capturing images of detected optical responses from a plurality of said cells.

WO 99/45357 PCT/US99/04473

13. The apparatus of claim 12 wherein said image capturing means comprises a charge coupled
device or CCD camera.

14. A method for detecting the response of individual cells to at least one
5 analyte of interest comprising:

a) providing a biosensor array comprising:

i) a substrate comprising a plurality of discrete sites; and

ii) a plurality of cells each dispersed at one of said discrete sites, wherein each cell is

encoded with at least one optically interrogatable material.
10 b) contacting said biosensor array with an analyte of interest; and

c) detecting an optical response of said ceils.

15. The method of claim 14 wherein

a) said providing a biosensor array comprises:

15 i) a substrate comprising a fiber optic array comprising a plurality of fibers, each fiber

having a distal end and a proximal end;

ii) said discrete sites comprise a plurality of microwells each formed on an end
surface of said distal end of said fibers;

iii) each one of said plurality of cells is dispersed in one of said
2 0 microwells; and

b) said detecting comprises providing a detecting means optically coupled to
and in optical communication with a proximal end of said fibers for detecting
an optical response of said cells at said distal fiber ends.

25 16. A method according to claims 14 or 15 wherein said analyte of interest
comprises an optically detectable label.

17. A method according to claims 14, 15, or 16 wherein said biosensor is contacted with a plurality of
analytes.

WO 99/45357 PCT/US99/04473

18. A method of making a biosensor array for detecting the response of
individual cells to at least one analyte of interest comprising:

a) providing a substrate comprising a plurality of discrete sites; and

b) contacting said substrate with a plurality of ceils sucht that each cell is dispersed at one of
said discrete sites.

19. A method according to claim 18 wherein

a) said providing a substrate comprises a fiber optic array comprising

i) a plurality of fibers, each fiber having a distal end and a proximal
10 end; and

ii) a plurality of microwells each formed on an end surface of said
distal end; and

b) said contacting comprises contacting said fiber optic array with a plurality
of cells such that each cell is inserted Into one of said microwells.

20 A method according to claims 18 or 19 wherein said plurality of cells
comprises at least two cell populations.

21 . A method according to claim 18, 19, or 20 wherein each ceil population is

2 0 encoded with at least one optically interrogatable material selected from the group consisting of a dye,
a fluorophore, a chromophore, a chemiluminescent compound and a bioluminescent compound.

22. A method according to claim 21 further comprising indexing the location of individual cells in said
array by emitted light energy produced by illuminating said encoded cells with excitation light energy.

WO 99/45357

PCT/US99/04473

1 / 10

Cells in Wells Concept

llllllll

III

Polished
Imaging Fiber

Microwells

Array of Cells

9 Cell Type #1
^ Cell Type #2
<§m Cell Type #3

Schematic concept of an optical sensor array of cultured cells. A polished optical imaging fiber is chemically
etched to produce an array of micron-sized wells at the distal l ace of the Fiber. Cells suspended in media above
the array of wells settle into the wells and adhere lo the well bottom. The wells are etched such that each well
accommodates one cell. Differences in core diameters and etch times can accommodate a wide variety of cell
types and sizes.

WO 99/45357

PCT/US99/04473

2 / 10

WO 99/45357

PCT/US99/04473

3 / 10

Cells in Wells Set-up

Capillary Tube
(1.5mm inner diameter)

Cell Suspension

Etched Optical Imaging Fiber
(7jim core diameter, 5jim core
depth, 1 mm fiber diameter)

Mounting Tack

Cells are deposited into wells at the distal face or an optical imaging fiber. Cells are trypsanized using aseptic
technique to remove iliem from the cell culture dish and suspended in growih media. A portion of the cell
suspension is concentrated using centrifugation and added to a 1 ,5mm diameter capillar,' tube. The etched tace
of the fiber id inserted into the tube and secured in place with a thin strip of laboratory film, the tube/fiber set-up
is then incubated in the vertical position for 12-15 hours for cell adhesion to occur.

WO 99/45357

4 / 10

PCT/US99/04473

WO 99/45357

5 / 10

PCTAJS99/04473

~~L=aT"L

BCECF-AM •

• »••-•» • •••

• • • • • *« • "

• •
• *

* i

Rinse

Schematic representation of the response of cells adhered in wells to the acetoxymethyl
(AM) ester form of the fluorscein derivative, bis-carboxyethyl bis-carboxy fluorscein
(BCECF). The array of cells (A) is incubated in a solution of BCECF-AM (B) and then
rinsed thoroughly. The non-fluorescent BCECF-AM passively enters the cell. Once
inside the cell, the lypophillic blocking groups are cleaved by the non-specific esteraces
resulting in an increase in fluorescent intensity (C).

WO 99/45357

6 / 10

PCT/US99/04473

£ ~ ^ i> c

= ~ 't = _

I I a* -£ « r ^ " > r

! *|J llfll

1 I * ! ^ HI

\ t j Z * -r * *T - 3 u

:-; J 13 2 5 S 5 S §

C _ — 1 'j a; t 5

C\ ■

—

-r

WO 99/45357

7 / 10

PCT/US99/04473

WO 99/45357

8 / 10

PCT/US99/04473

WO 99/45357 PCTAJS99/04473

10 / 10

Cell Viability in Etched Microwells

(average response of 25 cells)

Without Sonication

Time (min)

With Sonication (-15 min)

Time (min)

Live Wavelength (515nm)
Dead Wavelength ( 630nm)

pr^T w International Bureau

WORLD INTELLECTUAL PROPERTY ORGANIZATION
WORLU in international Bureau

(51) International Patent Classification * :
C12M3/04, 1/34

(11) International Publication Number: WO 99/45357

(43) international Publication Date: 10 September 1999 (10.09.99)

International Application Number:

(21)

(22) International Filing Date:

PCT/US99/04473
2 March 1999 (02.03.99)

(30) Priority Data:

09/033,462

2 March 1998 (02.03.98)

Hall, Medford, MA 02 1 55 (US).

(72) Inventors; and WALT David, R.

TAVXORTLaun, [US/US]; ISA Bradbury Avenue.
Medford, MA 02155 (US).
(74) Agent: CREEHAN. R.. Dennis; P.O. Box 750070, Arlington
( Heights. MA 02175^)070 (US).

ZW), Eurasian patent (AM AZ BY. KG BffiW £

55 CF.' CG, CI. CM. GA. GN. GW. ML. MR, NE.
SN.TD.TG).

Published

Z!XS2£*ZZ. UmU for amen d in g * cU-
££ trekked in the event of the receipt of amendments.

(88) Date of publication of the W^^XgW^I

(57) Abstract

sKssrssasssssssss-

FOR THE PURPOSES OF INFORMATION ONLY

Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.

Albania

Spain

Lesotho

Slovenia

Armenia

Finland

Lithuania

Slovakia

Austria

France

Luxembourg

Senega]

Australia

Gabon

Latvia

Swaziland

Azerbaijan

United Kingdom

Monaco

Chad

Bosnia and Herzegovina

Georgia

Republic of Moldova

Togo

Barbados

Ghana

Madagascar

Tajikistan

Belgium

Guinea

The former Yugoslav

Turkmenistan

Burkina Faso

Greece

Republic of Macedonia

Turkey

Bulgaria

Hungary

Mali

Trinidad and Tobago

Benin

Ireland

Mongolia

Ukraine

Brazil

Israel

Mauritania

Uganda

Belarus

Iceland

Malawi

United States of America

Canada

Italy

Mexico

Uzbekistan

Central African Republic

Japan

Niger

Viet Nam

Congo

Kenya

Netherlands

Yugoslavia

Switzerland

Kyrgyzstan

Norway

Zimbabwe

C6te d'lvoire

Democratic People's

New Zealand

Cameroon

Republic of Korea

Poland

China

Republic of Korea

Portugal

Cuba

Kazakstan

Romania

Czech Republic

Saint Lucia

Russian Federation

Germany

Liechtenstein

Sudan

Denmark

Sri Lanka

Sweden

Estonia

Liberia

Singapore

INTERNATIONAL SEARCH REPORT

tm tlonal Application No

PCT/US 99/04473

A. CLASSIFICATION OF SUBJECT MATTER

IPC 6 C12M3/04 C12M1/34

According to International Patent Classification (IPC) or to both national classification and IPC

B. FIELDS SEARCHED

Minimum documentation searched (classification system followed by classification symbols)

IPC 6 C12M

Documentation searched other than minimum documentation to the extent that such documents are inc

iuded in the fie

ds searched

Electronic data base consulted during the international search (name of data base and, where practical, search terms used)

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category •

Citation of document, with indication, where appropriate, of the relevant passages

Relevant to claim No.

FR 2 741 357 A (CORNING INC)

1-22

23 May 1997 (1997-05-23)

the whole document

2,5,6,9,

10,15,19

EP 0 539 888 A (SHIMADZU CORP)

1,3,4,

5 May 1993 (1993-05-05)

6-9,

12-14,

16,18,

20,22

figures 6,8

2,5,6,9,

10,15,19

WO 99 18434 A (TUFTS COLLEGE ; WALT DAVID R

1-13

(US); DICKINSON TODD A (US))

15 April 1999 (1999-04-15)

the whole document

-/—

Further documents are listed in the continuation of box C.

Patent family members are listed in annex.

• Special categories of cited documents :

"A* document defining the general state of the art which is not

considered to be of particular relevance
"E" earlier document but published on or after the international

filing date

"L" document which may throw doubts on priority claim(s> or
which is cited to establish the publication date of another
citation or other special reason (as specified)

"O" document referring to an oral disclosure, use, exhibition or
other means

"P" document published prior to the international filing date but
later than the priority date claimed

"T" later document published after the international filing date
or priority date and not In conflict with the application but
cited to understand the principle or theory underlying the
invention

"X" document of particular relevance; the claimed invention
cannot be considered novel or cannot be considered to
involve an inventive step when the document Is taken alone

"Y" document of particular relevance; the claimed invention

cannot be considered to involve an inventive step when the
document is combined with one or more other such docu-
ments, such combination being obvious to a person skilled
in the art.

document member of the same patent family

Oate of the actual completion of the international search

2 March 2000

Date of mailing of the international search report

09/03/2000

Name and mailing address of the ISA

European Patent Office, P.8. 5618 Patentlaan 2
NL - 2280 HVFHjswijk
Tel. (+01-70) 340-2040, Tx. 31 651 epo nl,
Fax: (+31-70) 340-3016

Authorized officer

Coucke, A

Form PCT7ISA/521 0 (second sheet) (July 1 992)

page 1 of 2

INTERNATIONAL SEARCH REPORT

Intt ionai Application No

PCT/US 99/04473

C.(Contlnuatlon) DOCUMENTS CONSIDERED TO BE RELEVANT

Category a Citation of document, with indication, where appropriate, ot the relevant passages

Relevant to claim No.

US 5 690 894 A (PINKEL DANIEL
25 November 1997 (1997-11-25)
cited in the application
claims; figures

ET AL)

2,5,6,9,
10,15,19

Forni PCT/ISA/210 (continuation of second sheet) (July 1992)

page 2 of 2

INTERNATIONAL SEARCH REPORT

information on patent family members

Into ional Application No

PCT/US 99/04473

Patent document

Publication

Patent family

Publication

cited In search report

date

member(s)

date

FR 2741357 A

23-05-1997

0862540 A

09-09-1998

9719027 A

29-05-1997

0539888

05-05-1993

5127099

25-05-1993

69210753

20-06-1996

69210753

02-10-1996

5348883

20-09-1994

9918434

15-04-1999

1269599

27-04-1999

5690894

25-11-1997

NONE

Form PCT/1&V210 (patent family annex) (July 1992)

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